Kinase Inhibitors and Nucleoside Analogues as Novel ...

Kinase Inhibitors and Nucleoside Analogues as Novel

Therapies to Inhibit HIV-1 or ZEBOV Replication

by

Stephen D. S. McCarthy

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Department of Laboratory Medicine and Pathobiology University of Toronto

© Copyright by Stephen D. S. McCarthy (2017)

ii

Kinase Inhibitors and Nucleoside Analogues as Novel

Therapies to Inhibit HIV-1 or ZEBOV Replication

Stephen D.S. McCarthy

Doctor of Philosophy

Graduate Department of Laboratory Medicine and Pathobiology

University of Toronto

2017

Abstract

Without a vaccine for Human Immunodeficiency Virus type 1 (HIV-1), or approved therapy for

treating Zaire Ebolavirus (ZEBOV) infection, new means to treat either virus during acute

infection are under intense investigation. Repurposing tyrosine kinase inhibitors of known

specificity may not only inhibit HIV-1 replication, but also treat associated inflammation or

neurocognitive disorders caused by chronic HIV-1 infection. Moreover, tyrosine kinase

inhibitors may effectively treat other infections, including ZEBOV. In addition, established

nucleoside/nucleotide analogues that effectively inhibit HIV-1 infection, could also be

repurposed to inhibit ZEBOV replication.

In this work the role of two host cell kinases, cellular protoncogene SRC (c-SRC) and Protein

Tyrosine Kinase 2 Beta (PTK2B), were found to have key roles during early HIV-1 replication in

primary activated CD4+ T-cells ex vivo. siRNA knockdown of either kinase increased

intracellular reverse transcripts and decreased nuclear proviral integration, suggesting they act at

the level of pre-integration complex (PIC) formation or PIC nuclear translocation. c-SRC siRNA

knockdown consistently reduced p24 levels of IIIB(X4) and Ba-L(R5) infection, or luciferase

iii

activity of HXB2(X4) or JR-FL(R5) recombinant viruses, prompting further drug inhibition

studies of this kinase. Four c-SRC kinase inhibitors (dasatinib, saracatinib, KX2-391 and SRC

Inhibitor-1) significantly reduced HXB2 and JR-FL infection in primary CD4+ T-cells. Thus,

these potent c-SRC inhibitors should be further evaluated in humanized mouse models of HIV-1

infection.

During 2014-16, the Ebola outbreak in West Africa prompted us to rapidly assess whether

conventional nucleoside analogs could inhibit in vitro ZEBOV replication. Employing a new

lifecycle model of ZEBOV infection in level 2 biocontainment, combinations of nucleoside

analogues and interferons were found to synergistically inhibit ZEBOV replication. These

included zidovudine, lamivudine and tenofovir, confirmed to show antiviral activity against fully

infectious ZEBOV-GFP in level 4 biocontainment. Findings from this thesis provided the

rationale for further preclinical development of nucleoside analogue combination treatments, and

a phase II EVD trial evaluating recombinant interferon in Guinea. Pre-clinical results using c-

SRC kinase inhibitors also suggest that this approach could also be effective in EVD.

iv

Acknowledgments

First and foremost, I will thank my mentor Dr. Donald R. Branch, for his inspiration, invaluable

advice and tremendous support throughout my time in his laboratory at Canadian Blood

Services. I also extend thanks to my Ph D advisory committee, Dr. Rupert Kaul and Dr. Dwayne

Barber, for their valuable advice, direction and input. I will next acknowledge the hard work of

Dr. Thomas Hoenen, Dr. Danila Leontyev, Dr. Anton Neschadim, Dr. Daniel Jung, Dr. Trina

Racine and Beata Majchrzak-Kita, who contributed immensely to the many joint projects related

to this work. As well, I am very grateful to Darinka Sakac, Yulia Petrenko and Amanda

Harrison Wong, who generously gave their time to train me, and Dr. Reed Siemieniuk for

insightful conversations. I also thank Dr. Eleanor Fish and Dr. Gary Kobinger for their

collaboration on a completely novel direction of this project, which has proven to be fruitful new

avenue of research.

I also thank my dear colleagues in the Branch laboratory, Evgenia Bloch, Carlyn Figueiredo,

Cindy Tong, Megan Blacquiere, Bonnie Lewis, Minji Kim, Eric Lai and Beth Binnington, for the

many opportunities to collaborate, abundant discussions, support of my ideas and work, and for

creating a joyful and lively lab environment. I also want to thank undergraduate summer

students Hannah Kozlowski, Shawn Goyal, Pauline Nicoletti, Ninon Guichard and Janette

Spears, whom I closely mentored and had good fortune to collaborate with over the years.

I also want to thank organizations that shaped my thinking around HIV and Ebola research. I am

eternally grateful for the support of my graduate department at the University of Toronto,

Laboratory Medicine and Pathobiology. In particular, I deeply appreciate the wisdom and

mentorship of Dr. Harry Elsholz, Dr. Maria Rozakis, Ferzeen Dharas-Sammy, Rama Ponda and

Katie Babcock, who encouraged my participation in the Three Minute Thesis competition, to

represent the department at the CIHR Canadian Student Health Research Forum in Winnipeg,

and to participate in our vibrant student union CLAMPS. Additionally, I am thankful to Sergio

Martinez, Duncan MacLachlan and Adam Busch, who at the AIDS Committee of Toronto taught

me valuable perspectives on people living with HIV-1, access to STI testing, and health care

inequality. I am also very thankful of Dr. Dana Devine and Don LaPierre at Canadian Blood

v

Services, who gave me a platform to advocate for blood donor equality at a national level. And I

am deeply thankful of Dr. Logan and Elizabeth Cohen, who through courageous actions,

reported potential alternative therapies to treat Liberian Ebola patients in 2014.

I will also take the opportunity to acknowledge the financial support of NSERC (CGS M

scholarship), Canadian Blood Services (GFP scholarship and support from the Centre for

Innovation to Dr. Branch), CIHR (Vanier CGS D scholarship, and support to Dr. Fish), the

Ontario HIV Treatment Network (Dr. Branch) and Health Canada (Dr. Branch), which have

financially supported me for the entirety of this project.

Finally, I would like to thank my dear parents, brothers and close friends. Your love and

unconditional support was the foundation of my success in graduate school.

vi

Table of Contents

Page Abstract ........................................................................................................................................... ii

Acknowledgments.......................................................................................................................... iv

Table of Contents ........................................................................................................................... vi

List of Tables ............................................................................................................................... viii

List of Figures ................................................................................................................................ ix

List of Abbreviations .................................................................................................................... xii

Chapter 1: Introduction ................................................................................................................... 1

1.1 HIV-1 and Ebola: Global Health Problems .......................................................................... 2

1.2 Human Immunodeficiency Virus (HIV) ............................................................................... 5

1.2.1 HIV-1 Epidemiology, Transmission and Replication Cycle .......................................... 5

1.2.2 HIV-1 Treatments, Potential Vaccines, and New Therapeutics ................................... 13

1.2.3 Host Kinases as Targets for HIV-1 Inhibition .............................................................. 17

1.2.4 The SRC Family of Non-Receptor Tyrosine Kinases in HIV-1 Infection ................... 22

1.2.5 Role of c-SRC in HIV-1 Infection ................................................................................ 37

1.2.6 Role of PTK2B in HIV-1 Infection .............................................................................. 42

1.3 Ebola Virus (EBOV) ........................................................................................................... 47

1.3.1 ZEBOV Epidemiology, Transmission and Replication Cycle ..................................... 47

1.3.2 ZEBOV Clinical Trials, Potential Vaccines and New Therapeutics ............................ 55

1.3.3 Repositioning Nucleoside Analogues for ZEBOV Inhibition ...................................... 59

1.3.4 Testing Other Potent Nucleoside/Nucleotide Analogues: Zidovudine, Lamivudine and

Tenofovir ............................................................................................................................... 69

1.4 Statement of the Problem, Rationale, Hypotheses and Objectives of this Work ................ 87

1.4.1 Statement of the Problem ............................................................................................. 87

1.4.2 Rationale ....................................................................................................................... 87

1.4.3 Hypotheses.................................................................................................................... 88

1.4.4 Objectives of this Work ................................................................................................ 89

1.4.5 Organization of the Thesis ............................................................................................ 89

vii

Chapter 2: c-SRC and PTK2B Protein Tyrosine Kinases Play Protective Roles in Early HIV-1

Infection of CD4+ T-Cell Lines. ................................................................................................... 91

2.1 Abstract ............................................................................................................................... 92

2.2 Introduction ......................................................................................................................... 93

2.3 Materials and Methods ........................................................................................................ 95

2.4 Results ............................................................................................................................... 101

2.5 Discussion ......................................................................................................................... 113

Chapter 3: c-SRC Protein Tyrosine Kinase Regulates Early HIV-1 Infection Post-Entry ......... 115

3.1 Abstract ............................................................................................................................. 116

3.2 Introduction ....................................................................................................................... 117

3.3 Materials and Methods ...................................................................................................... 119

3.4 Results ............................................................................................................................... 125

3.5 Discussion ......................................................................................................................... 140

Chapter 4: A Rapid Screening Assay Identifies Monotherapy with Interferon-ß and Combination

Therapies with Nucleoside Analogues as Effective Inhibitors of Ebola Virus .......................... 144

4.1 Abstract ............................................................................................................................. 145

4.2 Introduction ....................................................................................................................... 146

4.3 Materials and Methods ...................................................................................................... 148

4.4 Results ............................................................................................................................... 152

4.5 Discussion ......................................................................................................................... 169

Chapter 5: Key Findings, Future Perspectives, Conclusions and Broader Significance ............ 173

5.1 Key Findings, Limitations and Future Perspectives.......................................................... 174

5.2 Conclusions and Broader Significance ............................................................................. 189

References ................................................................................................................................... 195

Copyright Acknowledgments ..................................................................................................... 245

viii

List of Tables

Chapter 1

Table 1.1: Experimental drugs, therapeutics and vaccines fast-tracked for emergency II/III Ebola

clinical trials in 2014-2016. ............................................................................................................ 4

Chapter 4

Table 4.1: Fi and CI values for two-drug combination treatments. ............................................ 164

Table 4.2: Fi and CI values for three-drug combination treatments. .......................................... 165

ix

List of Figures

Chapter 1

Fig. 1.1: Global and national spread of HIV-1/AIDS. .................................................................... 6

Fig. 1.2: HIV-1 virion and genomic structure................................................................................. 8

Fig. 1.3: The HIV-1 replication cycle in T-cells. .......................................................................... 10

Fig. 1.4: FDA-approved kinase inhibitors. ................................................................................... 20

Fig. 1.5: Domain structure of chicken SRC-family kinases (SFKs). ............................................ 24

Fig. 1.6: Nef recycling of LCK and TCR impairs formation of the immunological synapse. ..... 27

Fig. 1.7: c-SRC domains, tertiary structure, and regulation. ........................................................ 38

Fig. 1.8: PTK2B domains, tertiary structure, and regulation. ....................................................... 43

Fig. 1.9: Spread of ZEBOV in West Africa during 2014-16. ....................................................... 48

Fig. 1.10: ZEBOV virion and genome structure. .......................................................................... 50

Fig. 1.11: The intracellular ZEBOV replication cycle. ................................................................. 52

Fig. 1.12: General structure of polymerases from the three main groups of RNA viruses and

retroviruses. ................................................................................................................................... 70

Fig. 1.13: Predicted structure of the RdRP L of ZEBOV. ............................................................ 72

Fig. 1.14: Model of ribonucleoside triphosphates in complex with RdRP L. .............................. 74

Fig. 1.15: Example nucleoside/nucleotide docking to the ZEBOV RdRP L. ............................... 76

Fig. 1.16: Nucleotide analogues binding ZEBOV RdRP L in the presence of NTPs................... 85

Chapter 2

Fig. 2.1: Western blot of phosphorylated proteins from serum-starved T-cells shortly after HIV-1

infection. ..................................................................................................................................... 102

Fig. 2.2: Jurkat T-cell lines inhibited with SRC-family kinase inhibitors. ................................. 104

Fig. 2.3: Cell survival was not significantly different between c-SRC drug or adenovirus vector

treatments. ................................................................................................................................... 105

Fig. 2.4: T-cell lines were transduced with adenovectors expressing c-SRC mutants. .............. 107

Fig. 2.5: siRNA titration and time-course optimization in Jurkat E6-1 cells. ............................ 109

x

Fig. 2.6: siRNA knockdown in Jurkat E6-1 cells infected with either VSV-G/HIV-1 or HXB2

virus............................................................................................................................................. 110

Fig. 2.7: qPCR of HXB2 cDNA extracted from Jurkat E6-1 following siRNA knockdown. .... 112

Chapter 3

Fig. 3.1: Cell Purity and Cell Viability of Enriched CD4+ T-Cells nucleofected with siRNA. . 126

Fig. 3.2: SRC-Family Drug Inhibitors do not Increase Necrosis or Apoptosis of Activated CD4+

T-Lymphocytes. .......................................................................................................................... 127

Fig. 3.3: Pre-treating CD4+ T-cells with c-SRC kinase inhibitors prior to HXB2 or JR-FL

infection. ..................................................................................................................................... 128

Fig. 3.4: c-SRC and PTK2B activation shortly after HIV-1 infection........................................ 130

Fig. 3.5: Luciferase reporter activity, viral integration and p24 production after c-SRC or PTK2B

siRNA knockdown. ..................................................................................................................... 132

Fig. 3.6: Cell surface receptor expression and RT activity. ........................................................ 134

Fig. 3.7: qPCR and PERT Assay Standard Curves. .................................................................... 136

Fig. 3.8: Time-Course and qPCR Melting Curves of HIV-1 DNA Targets. .............................. 137

Fig. 3.9: qPCR of early HXB2 or JR-FL infection after c-SRC or PTK2B siRNA knockdown.138

Fig. 3.10: qPCR of HXB2 or JR-FL Infection in CD4+ T-Cells from Three Separate Donors. . 139

Fig. 3.11: Summary schematic of the proposed mechanism for c-SRC involvement during early

HIV-1 infection. .......................................................................................................................... 140

Chapter 4

Fig. 4.1: Transcription and replication competent virus-like particle (trVLP) assay. ................ 149

Fig. 4.2: IFNs and nucleoside analogues inhibit Ebola mini-genome replication in vitro. ........ 153

Fig. 4.3: IFNs, toremifene, and nucleoside analogues inhibit trVLP replication. ...................... 154

Fig. 4.4: IFNs, toremifene and nucleoside analogues administered 24 hrs post-exposure inhibit

Ebola-mini-genome replication. ................................................................................................. 156

Fig. 4.5: No direct effect of drugs on Renilla luciferase reporter assay. .................................... 157

Fig. 4.6: CDV treatment increases trVLP replication. ................................................................ 158

xi

Fig. 4.7: IFNs, toremifene and nucleoside analogues reduce trVLP replication and transcription.

..................................................................................................................................................... 160

Fig. 4.8: 2 and 3 drug combinations synergistically inhibit Ebola trVLP infection. .................. 163

Fig. 4.9: IFNs, toremifene and nucleoside analogues administered 24 hrs post-exposure inhibit

ZEBOV-GFP............................................................................................................................... 167

Fig. 4.10: Synergistic 2 and 3 drug combinations against trVLP-LUC infection inhibit fully

infectious ZEBOV-GFP. ............................................................................................................. 168

Chapter 5

Fig. 5.1: Aberrant TCR signaling in Jurkat T-cells. ................................................................... 177

Fig. 5.2: Model of FPV-RTP and TFV-DP binding the RdRP L nucleotide pocket of ZEBOV.

..................................................................................................................................................... 187

xii

List of Abbreviations

(-)ssRNA negative-sense single-stranded RNA

(+)ssRNA positive-sense single-stranded RNA

1-LTR One Long Terminal Repeat

2ΔΔCT

Two delta delta Ct, comparative gene expression method

2-LTR Two Long Terminal Repeats

IIIB formerly HTLV-III B/H9, X4-tropic HIV-1

3P Triphosphate

3TC lamivudine, epivir

7-AAD 7-Aminoactinomycin D

α4β7 gut-specific homing integrin

Ab Antibody

ABL1 Abelson murine Leukemia viral oncogene homolog 1

ACK1 Activated CDC42 Kinase 1

ADV Adenovirus

AIDS Acquired Immunodeficiency Syndrome

ALT Alanine Transaminase

AMPK AMP-Activated Protein Kinase

ANP Acyclic Nucleotide Phosphonate

APC Antigen Presenting Cell

APC Allophycocyanin

APOBEC3G Apoliprotein B mRNA-editing Enzyme, Catalytic polypeptide-like 3G

ARG Abelson-Related Gene

ART Antiretroviral Therapy

ATCC American Type Culture Collection

ATP Adenosine Triphosphate

AZT Azidothymidine, zidovudine, retrovir

Ba-L Ba-Lung, R5-tropic HIV-1

BCR B-Cell Receptor

BCV Brincidofovir, CMX001

BCX4430 Immucillin-A

BDBV Bundibugyo Ebolavirus

BID Twice a Day

BLK B Lymphocyte Kinase

BMVEC Brain Microvascular Endothelial Cells

bNAB broadly Neutralizing Antibody

Bp Base Pair

BSL Biosafety Level

BVDV Bovine Diarrhea Virus

CA Capsid protein, p24

CADK Calcium-Dependent Tyrosine Kinase

cART combination Antiretroviral Therapy

CCL5 Chemokine (C-C motif) Ligand 5, RANTES

xiii

CCR5 Chemokine (C-C motif) Receptor 5

CD Cluster of Differentiation

CDC USA Centers for Disease Control and Prevention

CDV Cidofovir, CFV

cDNA complementary Deoxyribonucleic Acid

ChAd3-EBO-Z Chimpanzee Adenovirus type-3 vaccine vector expressing ZEBOV GP

CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

ChIP Chromatin Immunoprecipitation

CHK C-terminal SRC kinase-Homologous Kinase

CI Combination Index

CML Chronic Myelogenous Leukemia

CMV Cytomegalovirus

CNS Central Nervous System

Co-IP Co-Immunoprecipitation

CR3 Complement Receptor 3

CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR

associated protein 9

cRNA copy RNA

CRMP2 Collapsin Response Mediator Protein 2

CSF-1R Colony Stimulating Factor 1 Receptor

CSK C-terminal SRC Kinase

c-SRC cellular-SRC, proto-oncogene protein tyrosine kinase c-SRC, pp60c-SRC

CVID Common Variable Immunodeficiency

CXCR4 Chemokine (C-X-C motif) Receptor 4

c-YES cellular homolog of the Yamaguchi Sarcoma virus oncogene

DC Dendritic Cell

DC-SIGN Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-

integrin

DFP 4-amino substituted Diphenylfuropyrimidine

DISC Death-Inducing Signaling Complex

DMEM Dulbeco’s Modified Eagle’s Medium

DMSO Dimethyl sulfoxide

DN Dominant Negative

DNA Deoxyribonucleic Acid

DNAL4 DNA Ligase 4

dNTP deoxynucleoside Triphosphate

DP Dominant Positive

DP Diphosphate

ds DNA double-stranded Deoxyribonucleic Acid

DTT Dithiothreitol

DV Dengue Virus

DYRK1A Dual specificity tyrosine-phosphorylation-Regulated Kinase 1A

Early RT Early Reverse Transcripts

EBOV Ebolavirus

xiv

ECL Enhanced Chemiluminescence

EDTA Ethylenediaminetetraacetic Acid

EGFR Epidermal Growth Factor Receptor

ELISA Enzyme-Linked Immunosorbant Assay

EMD Emerin

Env Envelope

ERK Extracellular signal–Regulated Kinase

ESCRT Endosomal Sorting Complexes Required for Transport

ETC Ebola Treatment Center

EV Empty Vector

EV Enterovirus

EVD Ebola Virus Disease

EYFP Enhanced Yellow Fluorescent Protein

FACS Fluorescence-Activated Cell Sorting

FAK Focal Adhesion Kinase

FasR Fas Receptor

FAT Focal Adhesion Targeting domain

FBS Fetal Bovine Serum

FcγR Fc-gamma Receptor

FDA Food and Drug Administration (USA)

FERM Four point 1/Ezrin/Radixin/Moesin domain

FFU Focus-Forming Units

FGR Feline Gardner-Rasheed tyrosine kinase

FITC Fluorescein Isothiocyanate

FMDV Foot-and-Mouth Disease Virus

FPV Favipiravir, T-705

FSC Forward Scatter

FSV Fujinami Sarcoma Virus

FTC emtricitabine

FYB FYN Binding protein

FYN FGR- and c-YES-related protein kinase

Gag Group-specific antigen, polyprotein precursor Pr55

GAPDH Glyceraldehyde 3-Phosphate Dehydrogenase

GFP Green Fluorescent Protein

GM130 Golgi Matrix protein 130

gMFI geometric Mean Fluorescence Intensity

GORASP1 Golgi Reassembly-Stacking Protein 1

GP Glycoprotein

GP1,2 GP1 and GP2 glycoprotein

gp41 glycoprotein 41

gp120 glycoprotein 120

gp160 glycoprotein 160

GPCL cleaved Glycoprotein

GPCR G-Protein Coupled Receptor

xv

GRB2 Growth Factor Receptor-Bound protein 2

GS-5734 2-ethylbutyl l-alaninate phosphoramidate derivative

GTPase Guanosine Triphosphatase

HAART Highly Active Antiretroviral Therapy

HBV Hepatitis B Virus

HCK Hematopoietic Cell Kinase

HCV Hepatitis C Virus

HDAC Histone Deacetylase

HDP 3-hexadecyloxy-1-propanol

HEK 293T Human Embryonic Kidney cells 293 SV40 Large T-antigen

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer

HFV Human Foamy Virus

HIV-1 Human Immunodeficiency Virus type-1

HIV-2 Human Immunodeficiency Virus type-2

hnRNP-K heterogeneous nuclear Ribonucleoprotein K

HPC Hematopoietic Progenitor Cells

HRP Horseradish Peroxidase

HS Homo sapiens

HSV Herpes Simplex Virus

HTLV-1 Human T-cell Lymphotropic Virus type I

HXB2 HXB clone 2, X4-tropic HIV-1

IAV Influenza A Virus

ICAM-1 Intercellular Adhesion Molecule 1

IDR Intrinsically Disordered Region

IFN Interferon

IgG Immunoglobulin G

IL Interleukin

IL-2R Interleukin-2 Receptor

IN Integrase

IP Immunoprecipitation

IRF3 IFN Regulatory Factor 3

IS Immunological Synapse

ITAM Immunoreceptor Tyrosine-based Activation Motifs

JKC Jurkat C T-cells

JNK c-Jun N-terminal Kinase

JR-FL JR-Frontal Lobe, R5-tropic HIV-1

KNH1207 clinical isolate, R5-tropic HIV-1

KS Kaposi Sarcoma

Late RT Late Reverse Transcripts

LCK Lymphocyte-specific protein tyrosine Kinase

LDH Lactate Dehydrogenase

LEDGF Lens Epithelium-Derived Growth Factor, p75

LFA-1 Lymphocyte Function-associated Antigen-1

L-LEC Lung Lymphatic Endothelial Cells

xvi

LRA Latency Reversing Agents

LTR Long Terminal Repeat

LUC Luciferase

LYN LCK/Yes Novel protein tyrosine kinase

MA Matrix protein, p17

mAB monoclonal Antibody

MAPK Mitogen-Activated Protein Kinase

M-CSF Macrophage Colony-Stimulating Factor

MERS-CoV Middle East Respiratory Syndrome Coronavirus

MFI Median Fluorescent Intensity

MHC Major Histocompatibility Complex

MIP-1β Macrophage Inflammatory Protein 1 beta

MOI Multiplicity of Infection

MP Monophosphate

mRNA messenger Ribonucleic Acid

MS2 bacteriophage MS2

MSM Men who have Sex with Men

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

MVA-BN-Filo Modified Vaccinia Ankara expressing ZEBOV GP booster vaccine

MVC Maraviroc

NC Nucleocapsid, p7

Nef Negative effector

NF-κB Nuclear Factor and activator of transcription kappa B

NFAT Nuclear Factor of Activated T-cells

NHEJ Non-Homologous End-Join

NHP Non Human Primate

NIAID US National Institute of Allergy and Infectious Diseases

NNRTI Non-Nucleoside Reverse Transcriptase Inhibitor

NOD Non-Obese Diabetic

Nonidet P-40 octylphenoxypolyethoxyethanol

NoRT No Reverse Transcriptase

NP Nucleocapsid Protein

NPC1 Niemann-Pick C1

NRTI Nucleoside Reverse Transcriptase Inhibitor

N5SA Nonstructural protein 5A

N5SB Nonstructural protein 5B

NT Non-targeting siRNA

NTC No Template Control

NTP Nucleotide Triphosphate

P2Y2 P2Y purinoceptor 2

p38α mitogen-activated protein kinase 14

p53 tumor suppressor protein p53

PAK2 p21-Activated Kinase 2

PBMC Peripheral Blood Mononuclear Cell

xvii

PBS Phosphate Buffered Saline

PCR Polymerase Chain Reaction

PDGF-R Platelet-Derived Growth Factor Receptor

PE Phycoerythrin

PERT Product-Enhanced RT assay

PHA Phytohaemagglutinin

PHK Phosphorylase Kinase

PI3K Phosphoinositide 3-Kinase

PIC Pre-Integration Complex

PKC Protein Kinase C

PLC-γ1 Phospholipase C gamma 1

PMSF Phenylmethanesulfonyl Fluoride

Pol Polymerase

PP1 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine PP2 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine PPE Personel Protective Equipment

PR Protease

PREVAIL Partnership for Research on Ebola Vaccines In Liberia

PTK Protein Tyrosine Kinase

PTK2B Protein Tyrosine Kinase 2 Beta, FAK2, Pyk2

PTP Protein Tyrosine Phosphatase

PTP-PEST Protein Tyrosine Phosphatase Non-receptor type 12, PTPN12

PTPRA Receptor-type Tyrosine-Protein Phosphatase alpha, PTP-α

Q151Mc A62V, V75I, F77L, F116Y and Q151M complex

qPCR quantitative Polymerase Chain Reaction, real-time PCR

qRT-PCR quantitative Reverse Transcription Polymerase Chain Reaction

R5 CCR5-tropic HIV-1, M-tropic

Rac Rac subfamily of Rho small GTPases

rAD recombinant Adenoviral Vector

Ras Ras family of small GTPases

RdRP L RNA-dependent RNA polymerase L

RESTV Reston Ebolavirus

Rev Regulator of expression of virion proteins

RIPA Radioimmunoprecipitation Assay

RISC RNA-Induced Silencing Complex

RLU Relative Luciferase Units

RNA Ribonucleic Acid

RNAse H Ribonuclease H, p15

RPMI Roswell Park Memorial Institute medium 1640

RSK1 Ribosomal S6 Kinase 1

RSV Rous Sarcoma Virus

RT Reverse Transcriptase

RTC Reverse Transcriptase Complex

RTP Ribofuranosyl Triphosphate

xviii

RVFV Rift Valley Fever Virus

rVSV-ZEBOV-GP recombinant VSV vaccine expressing ZEBOV GP

Sam68 SRC-Associated substrate in Mitosis of 68 kDa

SAMHD1 SAM domain and HD domain-containing protein 1

SARS-CoV Severe Acute Respiratory Syndrome Coronavirus

SCID Severe Combined Immunodeficiency

SDF-1α Stromal cell-Derived Factor 1 alpha, CXCL12

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

SEM Standard Error of the Mean

SFK SRC Family of non-receptor tyrosine kinases

sGP soluble Glycoprotein

SH SRC Homology

SHP-1 Protein Tyrosine Phosphatase Non-receptor type 6, PTPN6

SHP-2 Protein Tyrosine Phosphatase Non-receptor type 11, PTPN11

siRNA small interfering RNA

SIV Simian Immunodeficiency Virus

SNALPS Stable Nucleic Acid Lipid Particles

SRC-I1 SRC Inhibitor-1

SSC Side Scatter

STAT1 Signal Transducer and Activator of Transcription protein 1

STI Sexually Transmitted Infection

SU6656 (3Z)-N,N-Dimethyl-2-oxo-3-(4,5,6,7-tetrahydro-1H-indol-2-

ylmethylidene)-2,3-dihydro-1H-indole-5-sulfonamide

SUDV Sudan Ebolavirus

SYBR Green nucleic acid staining dye

SYK Spleen-associated tyrosine Kinase

T-20 enfuvirtide

TAFV Taï Forest Ebolavirus

TAR Transactivation Response region

Tat Trans-activator of transcription, p16, p14

TBS Tris-Buffered Saline

TCID50 50% Tissue Culture Infective Dose

TCR T-Cell Receptor

TDF Tenofovir Disoproxil Fumarate

TEMED Tetramethylethylenediamine

Tev Tat, Env and Rev fusion protein

TFV Tenofovir, Viread

TGF-β Transforming Growth Factor beta

Th17 T helper 17

TIM-1 T-cell Immunoglobulin and Mucin Domain 1

TKM-Ebola Tekmira Pharmaceuticals siRNA treatment

TLR Toll-Like Receptor

TM DNA melting temperature

TNF-α Tumor Necrosis Factor alpha

xix

TOR Toremifene citrate

TP Triphosphate

Treg Regulatory T-cell

tRNA transfer Ribonucleic Acid

trVLP transcription and replication competent Virus-Like Particle

UCHT-1 anti-human CD3 antibody, clone UCHT-1

USAMRIID United States Army Medical Research Institute of Infectious Diseases

VACV Vaccinia Virus

VAV1 guanine nucleotide exchange factor 1

v-FPS viral FPS protein tyrosine kinase (avian)

Vif Viral infectivity factor

VMMC Voluntary Medical Male Circumcision

VP24 Viral Protein 24, matrix protein

VP30 Viral Protein 30, transcription activator

VP35 Viral Protein 35, polymerase cofactor

VP40 Viral Protein 40, matrix protein

Vpr Viral protein r

Vpu Viral protein u

Vpx Viral protein x

vRNA viral RNA

VS Virological Synapse

v-SRC viral-SRC

VSV Vesicular Stomatitis Virus

VSV-G Vesicular Stomatitis Virus Envelope G protein

VSV-G/HIV-1 VSV-G pseudoenveloped HIV virus

VZV Varicella Zoster Virus

WASp Wiskott-Aldrich Syndrome protein

WHO World Health Organization

WT Wild Type

X4 CXCR4-tropic HIV-1, T-tropic

ZEBOV Zaire Ebolavirus

ZMapp Mapp Biopharmaceutical monoclonal antibody cocktail

1

Chapter 1: Introduction

2

1.1 HIV-1 and Ebola: Global Health Problems

Notwithstanding decades of research in Human Immunodeficiency Virus type 1 (HIV-1) vaccine

design [1], prevention strategies to reduce transmission [2], or drug treatments to limit and

control infection [3], there are more than 36.7 million people living with HIV-1 worldwide as of

2015, and 1.1 million Acquired Immunodeficiency Syndrome (AIDS)-related deaths caused by

HIV-1 each year [4]. Another virus of global concern is Ebola virus (EBOV), which infected at

least 28,616 people and caused 11,310 deaths during the 2014-16 Zaire Ebola virus (ZEBOV)

outbreak in West Africa [5]. EBOV was discovered before HIV-1, yet there are no approved

antiviral agents to treat those infected [6]. The HIV-1 pandemic and EBOV outbreaks in Africa

in recent years have caused global shifts in how governments treat viral illnesses that

disproportionally affect the world’s poor, people who face stigma, and live in locations with

limited access to medications, trained medical staff or well-equipped hospitals. This has also

prompted changes in preclinical drug discovery research, to ultimately find effective treatments

that can prevent, cure or reduce chronic illnesses caused by either HIV-1 or ZEBOV, in novel

ways that can be implemented in resource limited settings or during a humanitarian crisis.

While treating HIV-1 infection in patients with combination antiretroviral therapy (cART) is

well tolerated and strongly suppresses patient viral load, these treatments are not without some

limitations [3, 7]. In the absence of a functional or sterilizing cure, the estimated 75,500

Canadians living with HIV-1 require life-time adherence to medications, which present new

challenges in reducing the rate of new HIV-1 infections [8]. Prolonged cART does not

significantly reduce the latent viral reservoir [3, 9], and there can be long-term toxicity

associated with sustained treatment with HIV-1 therapies [10-12]. In addition, some regimens

can cause unwanted drug-drug interactions in HIV-1 patients with comorbidities [13]. For

instance, the protease boosting agents ritonavir and cobicistat inhibit cytochrome P450-mediated

metabolism, altering the pharmacokinetics of co-medications [14, 15]. Thus there is a strong

incentive to discover novel therapeutics that can complement and improve current cART

medications.

3

Comparing to cART treatment of HIV-1, approved antiviral agents for treating Ebola Viral

Disease (EVD) are much further behind. Critical and supportive intensive care guidelines for

those suffering from EVD were still being developed during the 2014-16 ZEBOV outbreak in

West Africa, and continues to be an active area of research [16]. Critical care interventions

included: oral hydration therapy, intravenous fluid management to maintain blood electrolyte

balance, loperamide treatment for severe diarrhea, and pain relief [16]. Experimental drugs that

inhibit ZEBOV infection in vitro and in animal models of infection, which were safe and well-

tolerated in phase I clinical trials, became fast-tracked for emergency phase II/III trials that

began in 2014-15 (summarized in Table 1.1) [17-20]. These included trials assessing a cocktail

of three monoclonal antibodies called ZMapp [19], small-interfering RNA (siRNA) called TKM-

Ebola [21], transfusion of convalescent plasma [20] or recombinant interferon supplementation

[22]. Nucleoside analogues approved for treatment of other viruses that showed anti-ZEBOV

activity in vitro or in animal models of infection, such as favipiravir or brincidofovir, were also

considered for emergency phase II/III trials in West Africa [18, 23, 24]. Some of these clinical

trials are ongoing, in particular the PREVAIL vaccine trial in Liberia. Variability in non-

randomized trial design and limited patient enrollment produced modest support for any

particular treatment as the standard for treating EVD, while others were terminated before

efficacy could be assessed [25]. Success occurred with one vaccine trial, providing 100%

protection of those enrolled [26]. However treatment options for those with acute or chronic

EVD are much desired. Thus, continued preclinical research into novel ways to inhibit HIV-1

and ZEBOV replication is warranted.

4

Table 1.1: Experimental drugs, therapeutics and vaccines fast-tracked for emergency II/III

Ebola clinical trials in 2014-2016.

Medication Description Mechanism of

Action

Test species and

evidence Manufacturer

TKM-Ebola Small interfering

RNA (siRNA)

Targets ZEBOV

mRNA: RNA pol L,

matrix protein (VP24)

and polymerase

cofactor (VP35)

Protects cynomolgus

macaques [21]. Phase

II trial showed

therapeutic benefit

[27].

Tekmira

Pharmaceuticals

(Canada)

Brincidofovir

Lipid-conjugate

of cidofovir

(nucleoside

analogue)

Selectively inhibits

ZEBOV RNA-

dependent RNA

polymerase L

In vitro activity [28].

Phase II trial

terminated (low

enrollment) [24].

Chimerix (USA)

Favipiravir

Pyrazine-

carboxamide

derivative T-705

(nucleoside

analogue)

Selectively inhibits

ZEBOV RNA-

dependent RNA

polymerase L

In vitro, mouse model

of EVD [23, 29].

Phase II trial shows

efficacy in early

stages of EVD [18].

Toyama Chemical

(Japan)

ZMapp

Cocktail of three

monoclonal

antibodies

Binds viral envelope

GP1,2

Protects cynomolgus

macaques [30]. Phase

II trial showed

therapeutic effect, but

below statistical

threshold [19].

Mapp

pharmaceutical

(USA)

Interferon

(IFN) β-1a

AVONEX

(recombinant

interferon)

Promotes type 1IFN

response to counteract

VP24 and VP35

Prolongs macaque

survival [31]. Phase II

trial showed

therapeutic effect in

small cohort [22].

Biogen Inc. with

CIHR scientists

(Canada)

Vaccine Candidates

ChAd3-

EBOV-Z

Non-replicating,

recombinant

chimpanzee,

adenovirus type-

3 vector +

booster

Expressed the GP of

ZEBOV

Protects cynomolgus

macaques [32].

Phase II/III trial

results pending.

GlaxoSmithKline

(UK)

rVSV-

ZEBOV-GP

Live-attenuated,

recombinant,

vesicular

stomatitis virus

Expressed the GP of

ZEBOV

Protects cynomolgus

macaques [33].

Phase II/III trials

showed 100%

efficacy [26].

National

Microbiology

Laboratory

(Canada)

5

1.2 Human Immunodeficiency Virus (HIV)

1.2.1 HIV-1 Epidemiology, Transmission and Replication Cycle

HIV-1, the primary cause of AIDS, was first clinically observed in the United States in men who

have sex with men (MSM) in June of 1981 [34]. Otherwise healthy men succumbed to

pneumonia caused by Pneumocystis carinii, and developed the rare skin cancer Kaposi’s

sarcoma (KS) [34]. It was soon discovered patients were vulnerable to a variety of opportunistic

infections caused by bacteria, viruses and fungi [35], which were recognized as AIDS-defining

illnesses, permitting early classification of disease progression. In 1983-84, Dr. Robert Gallo,

Dr. Luc Montagnier and Francoise Barré-Sinousi were the first to report a new human T-

lymphotrophic retrovirus from AIDS patients [36, 37]. Shortly thereafter, Dr. Robert Gallo

independently demonstrated this new retrovirus to be the causative agent of AIDS [38, 39].

It has since been determined the HIV-1 pandemic started many decades prior to the first AIDS

cases identified in North America. Human-to-human transmission in Africa occurred as early as

the 1920’s in Kinshasa, the capital city of the Democratic Republic of the Congo (see Figure

1.1A, originally published in [4]). Early spread of HIV-1 from that era shares genetic similarity

with simian immunodeficiency viruses (SIVs) that infect the common chimpanzee Pan

troglodytes, suggesting HIV-1 originated as a zoonose from at least three separate cross-species

transmissions to humans [40]. Another serotype, HIV-2, is found predominantly in West Africa

and is associated with weaker transmission and less likely to cause AIDS, however co-infection

with HIV-1 can complicate antiretroviral treatment [41].

HIV-1 is found in a variety of bodily fluids, but is primarily transmitted in semen, vaginal

secretions or rectal secretions during intercourse, through transfusions of untreated blood

products, by reusing needles without sterilization, and by mother-to-child (vertical) transmission

from mixing of maternal and child blood at birth, or from breastfeeding [42]. Infant exposure to

maternal blood and fluids during childbirth is the most common route of vertical transmission.

In Canada, of the estimated 75,500 people living with HIV-1, 21% are unaware of their diagnosis

[8]. The 2014 incidence of new infections was 2,570, where MSM accounted for 54.3% of new

6

Fig. 1.1: Global and national spread of HIV-1/AIDS. (A) Adult prevalence of HIV-1 in

2015, based on UNAIDS country surveillance [4]. The highest prevalence of HIV-1 is in 8

southern countries of Sub-Saharan Africa. The star represents where HIV-1 originated in the

Democratic Republic of the Congo [43]. (B) The four primary modes of HIV-1 transmission in

Canada, as of 2014 [8]. (C) Declining global incidence of new HIV-1 infections or mortality

due to AIDS per year [4].

7

HIV-1 infections, followed by heterosexual intercourse (18.7%), people who emigrated from

HIV-endemic regions (13.9%), those of aboriginal ethnicity (10.8%) and injection drug users

(10.5%) (Figure 1.1B, originally published in [8]). Despite successes in reducing the global

levels of children born with HIV-1, HIV-1 testing of pregnant women, HIV-1 prevention

strategies, and increasing access to life-saving HIV-1 antiretroviral therapy, the global rate of

new infections remains persistently high at 2.1 million new infections in 2015, while HIV-1

mortality declines (Figure 1.1C, originally published in [4]).

HIV-1 is an enveloped retrovirus with a short 9.7 kb genome on positive sense, single-stranded

RNA ((+)ssRNA) (see Figure 1.2, originally published in [44] and [45]). The viral genome

contains three genomic regions (gag, pol and env) that encode genes that directly participate in

creating new virions, and 6 regulatory genes (tat, rev, nef, vif, vpr, and vpu). Together they

encode 19 distinct proteins [46]. The gag region encodes genes for the structural proteins p17

matrix (MA), p24 capsid (CA), p7 nucelocapsid (NC) and p6. The pol region encodes genes for

three viral enzymes: two subunits (p66 and p51) of reverse transcriptase (RT), integrase (IN) and

protease (PR), as well as p15 (RNase H). Finally, the env region encodes genes for the two

subunits of the external viral envelope protein, glycoprotein 120 (gp120) and glycoprotein 41

(gp41) [46]. The HIV-1 genome also encodes accessory proteins that regulate the viral lifecycle

and subvert immune responses to infection: negative regulatory factor (Nef), two splice variants

of trans-activator of transcription (p16 and p14 Tat), regulator of expression of virion proteins

(Rev), viral infectivity factor (Vif), viral protein u (Vpu), viral protein r (Vpr), and a fusion

protein encoded by tat, env and rev (Tev) [46].

During viral attachment and entry of a CD4+ T-cell, surface gp120, existing as a trimer of gp120-

gp41 heterodimers, binds to host CD4 [47]. Gp120 then binds a host chemokine co-receptor,

primarily α-chemokine CXCR4 or β-chemokine CCR5. CXCR4 is highly expressed on CD4+ T-

cells and CCR5 on the cell surface of macrophages, leading to the nomenclature of X4 viruses

(T-tropic) and R5 viruses (M-tropic), of which R5 viruses are more prevalent during early HIV-1

infection of a person and also more likely to be transmitted through intercourse [48, 49].

Following co-receptor engagement, virions that productively infect T-cells undergo

internalization by receptor-mediated endocytosis [50]. Within the endosome a conformational

8

Fig. 1.2: HIV-1 virion and genomic structure. (A) Schematic of a fully matured HIV-1 virion.

Transmembrane glycoprotein trimers, comprised of heterodimers of gp120-gp41, protrude from

the lipid membrane envelope [44]. Structural p17 Matrix (MA) protein associates with the

membrane envelope. The conical viral core is made of p24 capsid (CA) protein that surrounds

two strands of (+)ssRNA genome that interact with p7 nucleocapsid protein (NC), three

enzymes for replication (reverse transcriptase (RT), integrase (IN), and protease (PR)), as well as

viral accessory proteins (Vif, Vpr and Nef ). (B) Structure of the HIV-1 DNA genome with

flanking 5’ and 3’ long-terminal repeat (LTR) regions and three genomic regions (gag, pol and

env) that encode 19 distinct proteins through alternative splicing and cleavage by PR [45].

9

change in the glycoprotein trimer exposes the fusion domain of gp41, causing insertion of the

fusion peptide into the target T-cell membrane [51]. Inside the cytoplasm, the virus core makes

use of fibrous actin remodeling to dock outside the nucleus and shed capsid protein [52], creating

the reverse transcriptase complex (RTC) containing both viral and host proteins (Figure 1.3,

originally published in [53]). Viral reverse transcriptase synthesizes linear, double-stranded

copy DNA (ds cDNA) from the viral RNA genome, which binds integrase and other host factors

to form the pre-integration complex (PIC) [54]. The PIC is actively transported through a

nuclear pore and integrated into a host chromosome by reactions catalyzed by integrase and host

DNA repair proteins [55]. Failed integration of the cDNA are circularized by non-homologous

end joining, homologous recombination, or autointegration, leading to closed circular forms with

one or two long-terminal repeats (LTR), called 1- or 2-LTR circles [56]. Integrated provirus

behaves as a set of genes while 1- or 2-LTR circles are episomal DNA that may lead to

preintegration latency, a potential source of latent viral infection [57]. Expression of the

integrated virus is regulated by several host transcription factors that bind enhancer and promoter

sequences in the viral 5’ LTR, as well as viral Tat that binds the Transactivation Response region

(TAR) of nascent HIV-1 RNA transcripts in the nucleus, considerably increasing transcription of

the viral genome [58]. Unspliced and singly-spliced viral mRNA transcripts are exported into

the cytosol by Rev, then translated into proteins by the ribosome [59]. Separate from other viral

proteins, Env glycoprotein precursor (gp160) undergoes glycosylation and proteolytic processing

in the endoplasmic reticulum and trans-Golgi apparatus [60, 61]. Mature gp120-gp41

glycoproteins are then inserted into the host plasma membrane as non-covalently bound trimers.

Virus assembly at the plasma membrane is directed predominantly by Gag polyprotein precursor

(Pr55) [62] and the host actin cytoskeleton [63], leading to virions budding with encapsidated

genomic (+)ssRNA [64], viral enzymes, and host proteins located on the viral membrane and in

the virion itself [65]. Viral protease cleaves Gag and Pol polyproteins during the release of the

virus particle, creating fully mature virions capable of infecting new cells [66].

Following sexual transmission, dendritic cells (DCs), the most potent antigen-presenting cells

(APCs) of the adaptive immune system, play a key role in acquiring HIV-1 at mucosal surfaces

and disseminating the virus during early infection [67, 68]. Binding of virus to C-type lectin

10

Fig. 1.3: The HIV-1 replication cycle in T-cells. HIV-1 gp120 binds to host CD4 receptor, and

chemokine co-receptor CXCR4 or CCR5, which trigger lipid mixing and membrane fusion. The

viral nucleocapsid core, now in the cytoplasm, initiates reverse transcription of proviral ds cDNA

in the reverse transcriptase complex (RTC). The core uses actin filaments to dock outside the

nuclear pore, where CA protein sheds and IN forms the pre-integration complex (PIC) with the

cDNA. The PIC and host proteins transport the cDNA into the nucleus where it integrates into

host DNA by IN, or host DNA repair enzymes circularize non-integrated cDNA into 1-LTR or 2-

LTR circles. Integrated provirus can remain quiescent or be expressed by NF-κB or NFAT

initiating transcription. Multi-spliced viral mRNA are produced until enough Tat and Rev

protein cause unspliced mRNA to be synthesized and transported into the cytosol, forming the

full-length HIV-1 RNA genome. Viral proteins assemble with genomic RNA and bud as

immature virions, where PR activity cleaves proteins to create mature virions [53].

11

receptors on immature DCs, such as mannose receptor, langerin or Dendritic Cell-Specific

Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), leads to protection from

degradation in endolysosomal compartments [69, 70]. There, HIV-1 can remain infectious for 1-

3 days, providing enough time for immature DCs to migrate from the submucosa to local

lymphoid tissues and efficiently infect CD4+ T-lymphocytes [71]. Transmission occurs via three

routes: trans-infection through a virological synapse (VS) [72], exocytosis of virions from

multivesicular bodies [73], or by cis-infection from budding progeny virus [74]. HIV-1 can also

directly infect CD4+ T-cells at mucosal surfaces. In addition to CD4

+ T-cells, HIV-1 also infects

monocytes, macrophages and microglia cells [75], but predominantly infects and depletes the

helper CD4+ T-cell population during the course of infection [76, 77]. In a matter of weeks,

CD8+ cytotoxic T-cell and B-cell responses to control infection inadvertently provide immune

pressure that selects for the evolution of HIV-1 quasispecies [78]. Given the extensive genetic

diversity of HIV-1 in a host, virus expressing gp120 variants with different glycan shields can be

selected to evade clearance by broadly neutralizing antibodies [79]. Escape from broad humoral

responses results in persistent infection, causing depletion of Th17 CD4+ T-cells in gut mucosa

[80], mucosal damage from pro-inflammatory cytokines released from activated T-cells, and loss

of immune protection at intestinal mucosa [81]. Dysfunction at the intestinal barrier allows

microbial translocation of bacteria may contribute to systemic immune activation [82], which is

characteristic of chronic HIV-1 infection. This continued immune stimulation disrupts the

dynamic regulation of CD4+ and CD8

+ T-cells [83], leading to a gradual loss of peripheral CD4

+

T-cells over time from persistent viral replication and apoptosis [84, 85].

Chronic immune activation and inflammation also causes lymphoid tissue fibrosis via regulatory

T-cells (Treg) depositing collagen [86], and persistent antigen stimulation produces defective

helper T-cells that are less responsive, leading to effector T-cell exhaustion over many years

[87]. In addition, the immune system is unable to eliminate latently infected, central memory

CD4+ T-cells that produce low copies of virus, despite a patient adhering to suppressive therapy

and having an undetectable viral load [3]. Unfortunately, combination antiretroviral therapy

(cART) is unable to prevent the homeostatic proliferation of latently infected CD4+ cells that

harbor integrated provirus that are transcriptionally silent, nor access certain compartments of the

12

human body, such as the central nervous system, testes or gut-associated lymphoid tissue

(GALT), where low levels of viruses are produced and HIV-1 RNA and DNA are detected [88-

90]. These have been called anatomical sanctuaries, hypothesized to maintain the latent viral

reservoir through low-level viral replication in resting CD4+ T-cells, and are established within

days of primary infection [91]. Integrase inhibitors have been demonstrated to reach viral

sanctuaries such as the GALT, however they are unable to decrease the latent viral reservoir.

Thus, designing new antiviral combination therapies to activate infected, quiescent T-cells, then

clear infected cells after reactivation, has become a prominent area of HIV-1 cure research [92].

13

1.2.2 HIV-1 Treatments, Potential Vaccines, and New Therapeutics

In 1986-87, the first antiretroviral therapy (ART) approved for AIDS treatment in the United

States was the nucleoside analogue zidovudine (AZT) [93-95]. AZT, an inhibitor of HIV-1

reverse transcriptase, was swiftly used to treat AIDS patients at high doses to increase the CD4+

T-cell population and reduce opportunistic infections, but was associated with acute and long-

term drug toxicity such as anemia and neutropenia [96]. Moreover, drug resistance, as measured

by HIV-1 antigen detected in serum, developed within 24 weeks of continuous AZT treatment

[97]. It was also determined that HIV-1 contains an error-prone reverse transcriptase enzyme

that introduces base substitutions, additions and deletions when it synthesizes double-stranded

cDNA from the viral RNA genome [98]. These provide genetic variation in integrated provirus

that are selected under pressure of drug monotherapy, leading to escape mutations and the

evolution of drug resistant HIV-1 quasispecies [99-101]. In 1995, it was discovered that

combining AZT with lamivudine (3TC) was superior at controlling viremia when compared to

either monotherapy treatment [102]. This was superseded by triple drug therapies in 1996,

which considerably suppressed patient viral loads [103, 104]. The drug strategy was termed

highly active antiretroviral therapy (HAART), now called cART.

With 25 different single drugs and 14 fixed-dose combination regimens currently FDA-approved

to treat HIV-1, the success of cART has turned HIV-1 infection into a chronic, manageable

illness [7]. This paradigm shift has averted millions of AIDS-related deaths and controlled the

rate of new infections in developed and developing nations with the highest HIV-1 burden [105].

First-line therapy for treatment-naïve adults include three drugs, consisting of two nucleoside

reverse transcriptase inhibitor (NRTI) and an inhibitor from another drug class [106]. These

include non-nucleoside reverse transcriptase inhibitors (NNRTI), inhibitors that target viral

entry, fusion, integration or viral protease, and the potential inclusion of pharmacokinetic

enhancers [106]. While cART regimens can be well-tolerated and have a low pill burden for

patients, they require lifetime adherence to control viremia and any interruption can lead to rapid

viral rebound in plasma [107]. Drug interactions, unwanted side-effects, and regiment

adjustments can also lead to poor adherence among patients, which increase the risk of drug

14

resistance mutations that may cause virological failure of first-line cART [7]. The emergence of

multi-drug resistant HIV-1 variants has prompted renewed focus into novel vaccine and

therapeutic strategies to improve or replace cART [108], and ultimately end the AIDS pandemic

by developing a functional or sterilizing cure [109, 110].

There have been numerous attempts over the last 30 years to design a safe, immunogenic, and

effective vaccine to prevent or treat HIV-1 infection [1, 111]. These include active

immunization strategies to elicit broadly neutralizing antibodies (bNAbs) [112], inducing CD8+

T-cell responses [113], priming with Adenovirus (ADV) or canarypox vectors [114], or passive

immunotherapy with a human monoclonal antibody (mAb) [115]. After two decades of failed

vaccine trials, and one trial that lead to an increase in HIV-1 acquisition [116], the 2004-2009

RV144 trial in Thailand became the first vaccine to demonstrate efficacy in preventing 60.5% of

new HIV-1 infections after 1 year, and 31.2% after 3.5 years [1]. The prime-boost vaccine given

to 16,402 participants was a combination of two strategies: a cannarypox vector (ALVAC-HIV)

expressing clade E env and clade B gag and pro, and recombinant gp120 from clade B/E

(AIDSVAX). Participants that expressed IgG antibodies to the V2 loop of HIV-1 gp120 were

the least likely to become infected [1]. Further immunogenicity testing of the vaccine was

tested in South Africa in the 2013-14 phase I/II HVTN097 trial [117], providing the groundwork

for the phase II/III HVTN702 trial recently initiated in 2016. Therapeutic vaccines to intensify

cART treatment and limit establishment of the viral reservoir have produced few promising

leads, with the exception of the VRC01 monoclonal antibody isolated from the B-cells of an elite

controller [118]. Continued challenges remain in eliciting broadly neutralizing antibodies,

maintaining durable cross-strain breadth, and improving B-cell and T-cell priming to develop an

effective HIV-1 vaccine [108].

Other innovative strategies to prevent HIV-1 infection include male circumcision, preventative

microbicides and pre-exposure prophylaxis (PrEP). Since 2008, Voluntary Medical Male

Circumcision (VMMC) has successfully led to 11 million adolescent boy and adult male

circumcisions in eastern and southern Africa, as part of the ongoing World Health Organization

(WHO) strategy to prevent female-to-male HIV-1 infection [2]. To prevent male-to-female

infection, a variety of HIV-1 microbicides are being developed (acidic buffers, surfactants,

15

polyanionic polymers, reverse transcriptase inhibitors, and other small molecule inhibitors), with

different modes of delivery (gels, vaginal rings, tablets and nanoparticles) [119]. However

microbicides derived from cART regimens raise concerns of potential selection of drug-resistant

HIV-1 strains in women who seroconvert in microbicide trials [120]. In addition, cultural

expectations, partner-related factors (ex. fear of disproval) and acceptability issues have so far

led to low microbicide treatment adherence in clinical trials [121, 122]. In Canada, the recent

expansion of PrEP is a promising new strategy to prevent new HIV-1 infections, complementing

other prevention strategies such as access to HIV-1/STI testing and free condom distribution.

PrEP involves administering two ART medications, often tenofovir disoproxil fumarate (TDF)

with emtricitabine (FTC) in the single pill Truvada, to seronegative people who are at high risk

of contracting HIV-1 from a sexual encounter or the sharing of needles [123]. This can inhibit

HIV-1 before it disseminates from the site of infection at mucosal tissues, preventing systemic

infection [124]. A recent randomized, double-blind trial called IPERGAY determined that when

MSM participants were optimally adherent to PrEP, high plasma levels of antiretrovirals could

prevent 86% of new HIV-1 transmissions [125].

In terms of pre-clinical HIV-1 cure research, two main approaches are gene-editing to remove

integrated provirus, and latency reversal followed by clearing the viral reservoir. CRISPR/Cas9

technology is the leading gene-editing strategy to excise integrated HIV-1 provirus from the

primary CD4+ T-cells of HIV-1 patients ex vivo [126]. However, insertions and deletions that

follow Cas9 cleavage, created by host Non-Homologous End-Join (NHEJ) repair proteins, can

either suppress HIV-1 replication or accelerate viral escape by selection of viral sequences

refractory to Cas9 recognition [127]. Although a recent in vivo study has demonstrated proof-

of-principle for removing HIV-1 viral DNA in transgenic mice with CRISPR/Cas9 [109],

further research is needed to produce safe and efficient delivery of a mildly immunogenic viral

vector amendable for clinical use. Lastly, a ‘shock and kill’ strategy to reactivate then clear

latent HIV-1 infection, potentially curing a patient of HIV-1, involves pairing small-molecule

Latency Reversing Agents (LRAs), such as Histone Deacetylase (HDAC) inhibitors, with

immunotherapies that promote clearance of persistently infected cells [92]. Challenges remain in

reversing latency in all cell types harboring integrated provirus [128], boosting HIV-1-specific

16

CD8+ T-cell responses in tissue sanctuaries [129], and combining multiple LRAs in a

coordinated fashion with approaches to clear infection in animal models and human clinical trials

[130]. These complimentary approaches to replace or improve conventional cART are

promising areas of research. Yet there is also considerable interest in studying host T-cell factors

that contribute to HIV-1 replication, as novel means to restrict infection and eliminate the viral

reservoir of latently infected cells [131].

17

1.2.3 Host Kinases as Targets for HIV-1 Inhibition

Decades of basic HIV-1 research have revealed many complex interactions between HIV-1 and

the host immune cells that it infects. The success of FDA-approved HIV-1 antivirals targeting

host T-cell factors demonstrate both the critical nature of host proteins participating in the HIV-1

lifecycle, and their therapeutic value as components of cART [132, 133]. For example,

Maraviroc (MVC) was developed as a selective CCR5 receptor antagonist to block binding of

envelope gp120 [134]. Thus, discovering antivirals that target host factors essential for HIV-1

replication is a compelling area of research, as they may pose higher barriers to drug resistance

by blocking multiple stages of infection, offer unique tissue distribution to purge the viral

reservoir, or alleviate symptoms not targeted by conventional cART [135, 136].

With a genome encoding only 19 proteins, HIV-1 requires many host T-cell protein interactions

in order to replicate. These interactions have been organized into protein networks in an HIV-1

human protein interaction database [137]. As many as 348 unique protein-protein interactions

have been found between HIV-1 and human proteins in the Jurkat T-cell line [138]. Moreover,

mutations caused by the error-prone HIV-1 reverse transcriptase are primarily selected to negate

cellular restriction factors, exploiting host defense mechanisms that then improve viral fitness

[139]. As with other enveloped viruses, HIV-1 promotes replication and avoids immune

responses by modifying the host plasma membrane, which it also uses for its lipid shell during

budding. For instance, viral Nef and Vpu modulate plasma membrane receptor expression and

localization, to increase viral fitness and evade immune detection [140]. They can cause CD4

downregulation, which promotes viral egress by allowing newly synthesized gp120 to traffic to

the cell membrane, and inhibit tetherin binding, which allows newly released particles to bud

from the plasma membrane [140]. Alongside Vpu and Nef, HIV-1 encodes two other accessory

proteins (Vif, and Vpr) to counteract host restriction factors, each playing unique roles in

different T-cell types and at different stages of infection [141]. For example, host apolipoprotein

B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) is a cytidine deaminase

packaged into newly synthesized virions, catalyzing the deaminiation of cytosine shortly after

reverse transcription of nascent single-stranded viral cDNA in a new cell, dramatically impairing

18

HIV-1 replication [142]. Another host protein, SAM domain and HD domain-containing protein

1 (SAMHD1), can also interfere with reverse transcription by hydrolysing cytosolic

deoxynucleoside triphosphates (dNTPs) [143]. Yet these host proteins are counteracted by Vif,

which prevents APOBEC3G packaging into virions, and Vpx (the HIV-2 equivalent of Vpr),

which mediates degradation of SAMHD1 [141]. Moreover, viral enzymes such as IN depend on

host-cofactors to such an extent that drugs that interfere with integrase interactions with Lens

Epithelium-Derived Growth Factor (LEDGF/p75), called LEDGIN’s, show pre-clinical promise

as a new class of allosteric integrase inhibitors [144, 145]. Thus cellular proteins hijacked during

the HIV-1 replication cycle continue to be attractive targets in developing new antiretroviral

therapies.

In particular, HIV-1 modifies host signal transduction pathways that disrupt virtually every

aspect of cellular metabolism [146]. When gp120 binds to the co-receptor CXCR4 or CCR5

during viral entry of a T-cell, the interaction also initiates signaling pathways downstream to

promote intracellular viral replication post-fusion [147]. Corroborating this finding, CD4+ T-

cells isolated from asymptomatic, HIV-1 infected patients show defective early tyrosine

phosphorylation downstream of T-cell receptor stimulation [148, 149]. General inhibition of

tyrosine kinase signaling with the broad-spectrum inhibitor genistein has demonstrated that

tyrosine kinase signaling is essential for HIV-1 entry and intracellular steps shortly thereafter

[150, 151]. It has been put forth that HIV-1 requires T-cell activation and reorganization of the

cytoskeleton for productive HIV-1 entry and replication [152], altering tyrosine kinase pathways

downstream of the T-cell Receptor (TCR) to facilitate its viral lifecycle. In support of this

hypothesis, the addition of mitogenic stimuli can activate host kinases to permit HIV-1

replication in quiescent T-cells that is otherwise inefficient, further suggesting a potential role of

host T-cell kinases in the HIV-1 lifecycle [153]. As mentioned earlier, chronic T-cell activation

also has an important role in maintaining persistent HIV-1 infection, helper T-cell disregulation,

and eventual exhaustion of effector T-cells [83, 87] further emphasizing the important roles of

tyrosine kinase signal transduction.

Small lentiviruses, such as HIV-1, appear to have evolved to become phosphorylated by host T-

cell kinases to facilitate infection in non-dividing cells, access the nuclear compartment, and

19

assemble proteins during viral egress [154]. All HIV-1 proteins are phosphorylated throughout

intracellular viral replication, however the functional purpose for these modifications, and the

kinases that regulate them, is an active area of research [155]. Thus far, phosphorylation events

have been found to regulate: HIV-1 viral entry [147], actin remodeling [52, 135], reverse

transcription [156], capsid shedding [157], nuclear translocation of HIV-1 ds cDNA [154, 155,

158], viral integration [159], post-integration repair and circularization of un-integrated cDNA

[160], proviral gene transcription [161], mRNA transport [162], viral assembly [63], and budding

from the host T-cell [163]. One of the most well studied HIV-1 proteins that modify cellular

tyrosine kinase activity is Nef, which has pleiotropic effects on cell signaling and strong binding

affinity to the SRC family of kinases (SFKs) [164-167]. Nef preferentially activates SFK

members LCK, HCK, LYN and c-SRC through allosteric displacement of intramolecular SRC

homology 3 (SH3)-linker interactions [164], however its functional role in activating SFKs

during HIV-1 replication is still under investigation, such as the SFK-Nef contribution to AIDS

progression [168].

Currently there are 28 small-molecule kinase inhibitors approved by the US Food and Drug

Administration for various cancer indications (Figure 1.4, originally published in [169] and

[170]). Their improved specificity from first generation inhibitors and known inhibition of host

kinases has made them ideal for other illnesses outside of cancer, such as pulmonary fibrosis

[171], arthritis [172], and potentially viral infections [173]. Unfortunately, HIV-1 patients taking

cART are at higher risk for non-AIDS defining cancers such as lung adenocarcinoma, Hodgkin’s

lymphoma and anal cancer, when compared with the average population [174]. It has been

suggested this increased risk in due to heightened immune activation and inflammation [175].

Repositioning safe and well-tolerated kinase inhibitors, with published safety and efficacy for

various cancer indications, offers the potential for a new class of antivirals to inhibit HIV-1. In

particular, tyrosine kinase inhibitors are attractive for these purposes because of

20

Fig. 1.4: FDA-approved kinase inhibitors. (A) The cancer and non-cancer indications of

small-molecule kinase inhibitors as of 2015. Dasatinib, a dual ABL/SRC inhibitor that has

shown antiviral activity, is highlighted in red [169]. (B) Chemical structure of dasatinib, with

emphasis of its mode of binding to the adenine pocket and hydrophobic pocket of ABL kinase

[170]. (C) Co-crystal structure of dasatinib (purple) in the Adenosine Triphosphate (ATP)

pocket of ABL, interacting with key amino acids residues (green) through hydrogen bonding (red

dashed lines) [170].

21

extensive research into their molecular interactions with host proteins, known off-targets, ease of

administration, biodistribution, and pharmacokinetics within humans [169, 170]. Some of these

inhibitors, such as imatinib, have been used successfully to treat Kaposi Sarcoma lesions and

Chronic Myeloid Leukemia (CML) in HIV-1 patients on cART [176, 177]. Others have shown

antiviral activity during virus replication, such as the dual SRC/ABL inhibitor dasatinib,

restricting Dengue Virus (DV) replication in vitro [178]. Moreover, extended use of drugs that

inhibit HIV-1 directly are prone to selection of drug-resistant variants, whereas small-molecule

inhibitors targeting host factors may pose a greater mutational barrier, reducing the chance of

selecting drug-resistant viral strains [152]. Thus novel kinase inhibitors that reduce

inflammation, target cancer, and restrict HIV-1 replication, would be ideal drug candidates to

improve cART regimens.

In the human genome there are 32 non-receptor tyrosine kinases (NRTKs) that catalyse the

phosphorylation of tyrosine residues on protein substrates, grouped into 10 families [146].

NRTKs regulate cell processes essential to life such as cell signaling, growth, differentiation,

motility, adhesion and cell death [179-183]. While NRTKs are generally appreciated for having

central roles in cancer and chronic inflammation [184], they are increasingly being recognized

for playing significant roles during viral infections. However, there are specific gaps of

knowledge stalling FDA-approved tyrosine kinase inhibitors from being repurposed to treat

HIV-1 in conjunction with cART. In the following three sections, the role of SRC family

kinases and focal adhesion kinases (FAK) during HIV-1 infection will be reviewed.

22

1.2.4 The SRC Family of Non-Receptor Tyrosine Kinases in HIV-1 Infection

In 1911, Dr. Francis Peyton Rous observed that a virus in cell-free filtrate can cause

fibrosarcoma cancer in domestic chickens [185], latter named the Rous Sarcoma Virus (RSV).

For this discovery of tumor-inducing viruses, Dr. Rous was awarded the Nobel Prize in

Physiology or Medicine in 1966. This soon led to the search for the oncogene responsible for

transformation caused by retroviruses. Drs. John Michael Bishop and Harold Eliot Varmus

demonstrated in 1976 that the viral oncogene responsible for avian sarcomas was originally

acquired from normal avian cells [186]. The viral oncogene causing sarcomas was called v-

SRC, to distinguish it from the cellular homolog c-SRC. Drs. Bishop and Varmus received the

1989 Nobel Prize in Physiology or Medicine for their work showing the cellular origin of

retroviral oncogenes, and that a malignant tumor can originate from normal genes that regulate

growth within a cell. Then at the University of British Columbia in 1981, while investigating

Fujinami Sarcoma Virus (FSV) regulation of v-FPS signaling, Dr. Anthony James Pawson made

an important breakthrough as he uncovered the first modular domain that controls cellular signal

transduction [187]. He identified a phospho-tyrosine binding site similar to a non-catalytic

region of v-SRC, called the SRC homology 2 or SH2 domain, which became prototypic of other

non-catalytic modules that modify kinase activity allosterically, or through binding to other

signaling proteins [188]. Since these pioneering discoveries, hundreds of modular protein

domains have been identified that underpin multiprotein signaling complexes. Much has also

been learned of the structure, regulation, localization and function of v-SRC, c-SRC, and the

related SRC family of non-receptor tyrosine kinases [189].

In humans, there are eight SFK members: c-SRC, LCK, FYN, HCK, LYN, FGR, c-YES and

BLK [189]. The 52-62 kDa proteins play essential roles in the signaling of a variety of cell

processes, such as proliferation, differentiation, motility, adhesion, and proper functioning of

adaptive and innate immunity [189-191]. c-SRC, FYN and c-YES show ubiquitous expression

in many cell types and tissues, while the remaining five have time-specific and cell lineage-

specific expression, such as in hematopoietic cells [192-196]. SFKs are activated by the

stimulation of a variety of transmembrane G-protein coupled receptors (GPCRs), such as the C-

23

X-C chemokine receptor type 4 (CXCR4) and C-C chemokine receptor type 5 (CCR5), linking

surface receptors with downstream signaling pathways such as Mitogen-Activated Protein

Kinase (MAPK) activation [197]. SFKs have seven conserved domains that regulate their

function (see Fig. 1.5 for their domain structure, originally published in [198]). The N-terminus

contains a 15 amino-acid peptide sequence that forms the SH4 domain, involved in post-

translational lipid modifications of the SFK such as myristoylation or palmitoylation [199].

These modifications target SFKs towards membrane-associated signaling and adaptor protein

complexes in various cell compartments, or lipid rafts positioned at the inner leaflet of the

plasma membrane. The SH4 domain is followed by a unique region of 50-90 residues that is

characteristic to each SFK [199]. This intrinsically disordered region (IDR) can encode

sequences that promote cleavage of the SFK protein, phosphorylation of residues that add further

specificity to kinase activity, and localization signals. After the IDR is an SH3 module that

mediates intra- and intermolecular protein binding by recognizing the proline-rich PxxP

consensus sequence. Next there is an SH2 domain capable of binding proteins with

phosphorylated tyrosine in a pYEEI consensus sequence [199]. A short SH2-linker region

containing proline-rich sequences follows the SH2 domain, a target region for SH3 binding.

This linker region is followed by a large SH1 domain containing the catalytic site for substrate

and ATP binding, and an activation loop where the trans- or autophosphorylation of a specific

tyrosine residue fully activates the kinase [199]. Lastly, there is a short, negative regulatory C-

terminus tail with a terminal tyrosine residue that when phosphorylated, binds the SH2 domain.

While healthy cells tightly regulate the active and inactive conformations of each SFK, viruses

have been implicated in altering their expression, activity or cellular localization [178, 200].

These can impair innate or adaptive immune responses to clear viral infection, promote spread of

the virus within tissues or between cells, prevent apoptotic cell death of infected cells, or directly

assist in the viral replication cycle [201]. For instance, cellular SFKs facilitate the entry of the

coxsackievirus, the RNA replication of Hepatitis C Virus (HCV) and DV, as well as assembly of

West Nile Virus (WNV) [178, 201-203]. The multiple mechanisms by which the SRC-family of

kinases facilitate or impede the infectious cycle of HIV-1 will be examined in this section,

suggesting potential roles for c-SRC interactions with HIV-1, reviewed further in chapter 1.2.5.

24

Fig. 1.5: Domain structure of chicken SRC-family kinases (SFKs). (A) Linear representation

of the seven domains characterisitic of all SFKs: C-terminus tail with a negative regulatory Tyr

residue, kinase domain (SH1), proline-rich SH2 linker, a domain that binds phosphorylated Tyr

(SH2), a domain that binds proline-rich motifs (SH3), a short unique region, and an N-terminus

that can be post-translationally modified (SH4). (B) Phosphorylated Tyr527/530 binds SH2,

bringing the SH3 domain in close proximity of the SH2-linker, holding the SFK in an inactive

conformation. Dephosphorylation of Tyr527/530 and phosphorylation of Tyr416/419 activate

the enzyme. (C) General mechanism of receptor-mediated SFK recruitment leading to full SFK

activation [198].

25

LCK

Lymphocyte-specific protein tyrosine kinase (LCK) is expressed in B-cells, T-cells, natural killer

cells, brain tissue and in the spleen [204-207]. It localizes to the plasma membrane and to

pericentrosomal vesicles in the cell, interacting with the cytoplasmic tails of transmembrane

receptors [208]. LCK non-covalently associates with CD4 or CD8, plays a major role in TCR-

CD3 mediated T-cell activation, and is a downstream signaling molecule of the interleukin-2 (IL-

2) receptor [209, 210]. In CD4-expressing T-cells, 50-96% of LCK is bound to the cytoplasmic

tail of CD4 receptors, through interactions between the N-terminus of LCK and cysteine residues

on CD4 [211]. Cross-linking of CD4 on the cell surface transiently increases the enzyme activity

of LCK, and it is believed autophosphorylation of Tyr394 participates in this increased LCK

activation [212]. In JCaM1 T-cells expressing a defective splice variant of LCK, cells are unable

to respond to TCR-CD3 stimulation [213]. However, expression of wild type LCK restored TCR

signaling, demonstrating the significance of LCK in normal TCR-CD3 signaling pathways [213].

In the CD4+ T-cells isolated from chronically infected HIV-1 patients, these cells show defective

responses to CD3 activation, reduced proximal TCR tyrosine phosphorylation, and decreased

levels of LCK expression [148]. Paradoxically, acute infection of Jurkat T-cells with HIV-1IIIB

show a global increase in tyrosine phosphorylation within 30 minutes, including activation and

phosphorylation of LCK [214]. Gp120/CD4/LCK complex signalling can induce NF-κB nuclear

translocation and gene transcription [215]. These studies suggest nuanced and different effects

of LCK throughout HIV-1 infection, which appears to be protective during early entry as LCK

associates with CD4. For example, point-mutations of LCK at Cys20 and Cys23, which prevent

LCK association with CD4, results in greater HIV-1 replication [209]. Moreover JCaM1.6 T-

cells, expressing truncated LCK, are far more infectable than Jurkat 45 cells, which lack CD45

and express much higher LCK activity [209]. It has also been demonstrated that short-term

exposure of gp120 can transiently enhance the autophosphorylation and activity of LCK, while

long-term incubation of gp120 with CD4+

T-cells decreases overall expression of LCK [148,

216]. This longer gp120 exposure also correlates with complete dissociation of LCK from CD4,

followed by downregulated surface expression of CD4 [217]. Furthermore, HIV-1 gp120 can

block the migration of CXCR4-expressing T-cells to stromal cell-derived factor 1 alpha (SDF-

26

1α), which is mediated by CD4/LCK signaling and associated with cofilin phosphorylation

[218]. The precursor protein, gp160, can also disrupt adhesion between CD4+ T-cells and B-

cells by donwregulating lymphocyte function-associated antigen-1 (LFA-1), in an LCK-

dependent manner [219].

The use of a variety of techniques to overexpress or knockdown LCK, coupled with

colocalization experiments, have revealed many of the mechanisms by which HIV-1 proteins, in

particular Nef, Vpu and Tat, interact with LCK to modify its initial protective function in order

to facilitate viral replication. In transgenic mice expressing HIV-1 Nef, the animals exhibit

profound thymocyte depletion and CD4+ T-cell lymphopenia [220]. It has been found that Nef

depletes the population of double-positive thymocytes and impairs lineage commitment towards

single-positive CD4+ thymocytes, which was dependent on reduced LCK activity [220].

Expressing constitutively active LCK in the mice rescued CD4+

T-cell maturation, potentially

through increased affinity of the TCR- Major Histocompatibility Complex (MHC) interaction

[220]. HIV-1 Nef disrupts the anterograde transport of LCK to the plasma membrane within T-

cells [167]. It does this by interfering with the vesicular transport of newly synthesized LCK to

membrane-microdomains, a mechanism dependent on the SH4 membrane-anchor domain of

LCK [167]. Nef also impairs the formation of the immunological synapse (IS) and early T-cell

signaling, by retargeting the TCR and LCK for recycling in endosomal compartments (see Figure

1.6, originally published in [221]). It induces the internalization and endocytosis of surface CD4

in a manner dependent on LCK in lymphoid cells [222]. In addition, Nef relocalizes LCK to the

trans-Golgi network, effectively decoupling downstream LCK kinase signaling from the surface

TCR-CD3 complex [223]. In this way, Nef diminishes TCR responses to antigenic stimulation

while rerouting LCK and other kinases to the trans-Golgi network, promoting T-cell survival

through IL-2 production and enhancing viral spread of infected cells [223]. Similar to Nef, the

viral accessory protein Vpu can also subvert the subcellular localization of LCK, implicating

LCK with Vpu-mediated downregulation of cell surface receptors [224]. Nef also recruits a

variety of proteins, including heterogeneous nuclear Ribonucleoprotein K( hnRNP-K) and LCK,

into a signaling complex that increases Tat-mediated HIV-1 transcription [225]. This

27

Fig. 1.6: Nef recycling of LCK and TCR impairs formation of the immunological synapse.

(A) In resting T-cells, LCK and T-cell receptors (TCRs) continuously traffic through early

endosomes. Upon TCR engagement with an antigen, polarized recycling targets TCRs and LCK

to the immunological synapse, inducing T-cell activation. (B) During HIV-1 infection, viral Nef

strongly retains LCK in recycling endosomes. TCRs also accumulate in the early endosome

from reduced trafficking to the plasma membrane. Both of these effects lead to inefficient

targeting of LCK or TCR to form an immunological synapse upon antigen stimulation [221].

28

interaction offers one potential explanation of how Nef enables high levels of viral replication in

HIV-1 infected people. In addition, LCK has been shown to participate in Tat-mediated

induction of NF-κB reporter gene expression in Jurkat T-cells [226].

LCK kinase activity also influences viral egress and cell-to-cell spread of HIV-1. Activated

LCK directly interacts with Gag protein through its unique domain in infected cells, and

facilitates viral assembly at the plasma membrane [227]. It was determined that palmitoylation of

the unique domain of LCK was required for the successful release of HIV-1 virus-like particles

[227]. In a phosphoproteome study of signaling pathways essential for virus cell-to-cell spread,

LCK and other distal pathways of the TCR were activated and found to be essential for viral

dissemination by the virological synapse [228]. Taken together, LCK initially promotes

protection against HIV-1 infection downstream of CD4 and TCR-CD3 engagement during acute

infection, becomes decoupled from CD4 and relocalized to the trans-Golgi complex where it

facilitates downregulation of cell surface receptors, assists with viral egress and viral spreading,

and becomes downregulated during chronic HIV-1 infection.

FYN

Similar to LCK, the FGR- and c-YES-related protein kinase (FYN) is involved in TCR-CD3

signaling in CD4+ and CD8

+ T-cells, as well as IL-2 production [149, 229]. FYN also

participates in Central Nervous System (CNS) myelination during neuronal development and the

formation of a variety of cancers [230, 231]. FYN can be found in many cell types, although a

unique isoform of FYN, called FYN-T, is expressed only in hematopoietic cells and contributes

to TCR-induced calcium fluxes of antigen-stimulated T-cells [232]. It participates in integrin

signalling, associates with FYN binding protein (FYB) to modulate IL-2 expression, and innate

immune signaling through Toll-Like Receptors (TLRs) expressed on T-cells [233-235].

In the CD4+ and CD8

+ T-cells from HIV-1 non-progressors, FYN is super-activated compared

with cells collected from uninfected controls or HIV-1 patients with AIDS symptoms [149].

This points towards a potential protective mechanism associated with FYN kinase activity.

Corroborating this finding, S1T cells, which lack LCK and FYN expression, are more infectable

29

by HIV-1 [209]. Similar to LCK, pre-treating cells with HIV-1 gp160 prevents CD3-mediated

activation of CD4+ T-cells and FYN phosphorylation, demonstrating that gp160/CD4 signaling

also deregulates the TCR-CD3 activation of FYN [236]. Also similar to LCK, acute HIV-1IIIB

infection of Jurkat T-cells activates FYN activity in the first 30 minutes of infection [214].

Furthermore, FYN can activate the transcription of the HIV-1 promoter by activating four NF-κB

DNA-binding proteins in Jurkat T-cells [237]. This activity required the SH2 domain of FYN

[237]. Unlike LCK however, the effect of FYN during HIV-1 infection is not through an

interaction with Nef. While Nef can bind the isolated SH3 domain of FYN in vitro, it does not

bind full-length FYN, nor has a direct effect on FYN kinase activity in vivo [164].

During the HIV-1 lifecycle, FYN has been shown to directly interact with viral Vif and promote

virion assembly and release [227, 238]. In the absence of Vif, activated FYN is able to

phosphorylate the cytosine deaminase APOBEC3G, increasing the level of phosphorylated

APOBEC3G becoming encapsidated into budding virus, which can restrict viral replication in

the cytosol in a subsequent target T-cell [238]. However in the presence of Vif, Vif protein

binds the SH3 domain of FYN, reducing FYN tyrosine kinase activity. Vif also reduces the

autophosphorylation of FYN [238]. This reduced FYN activity leads to less phosphorylation of

APOBEC3G packaged into virions, counteracting this natural HIV-1 resistance factor in

successive infection cycles and promoting viral infectivity [238]. Interestingly, palmitoylated

FYN can enhance the assembly and release of HIV-1 virus-like particles, in a similar manner as

LCK [227]. This was also dependent on the N-terminal sequence of FYN, localizing FYN to

sites of virus budding at the plasma membrane [227]. Thus FYN activity has multiple effects on

HIV-1 during the viral replication cycle, with different signaling functions as HIV-1 enters the

cell, initiates viral transcription, packages viral and host proteins into virions, and buds from an

infected cell.

HCK

Hematopoietic cell kinase (HCK) is primarily expressed in B-lymphoid and myeloid cell

lineages, such as promoncytic cells and monocyte-derived macrophages, and is not expressed in

CD4+ T-cells [239]. In phagocytic cells, HCK mediates proinflammatory cytokine production,

30

Fc-gamma receptor 1 (FcγRI) signaling, phagocytosis, migration and cell spreading [240-242].

It also has important functions in the migration and degranulation of neutrophils [243].

In primary, naive B-cells, it has been found that HIV-1 gp120 signals through integrin α4β7,

resulting in reduced proliferative responses that correlated with downregulated expression of

HCK [244]. This may contribute to the delayed and ineffective humoral response after acute

HIV-1 infection, as these primary B-cells treated with gp120 also increased expression of the

immunosuppressive cytokine TGF-β1 [244]. After B-cells, HCK is most strongly expressed in

monocytes/macrophages, which are important target T-cells of HIV-1 replication and

maintenance of the viral reservoir. In monocyte-derived macrophages stimulated by macrophage

colony-stimulating factor (M-CSF), high HCK expression correlated with increased HIV-1

infection, and reducing HCK expression with antisense oligonucleotides blocked viral replication

[245]. Of all the SFKs, viral Nef most strongly binds HCK, and this interaction has been

described in great detail [164]. Nef activates HCK in vitro and in primary macrophages, and the

Nef PxxP motif is essential for Nef binding to the SH3 domain of HCK [164]. This implies that

Nef activates SFKs through intramolecular displacement of SH3 binding to the SH2-linker.

Interestingly, dephosphorylation of the negative regulatory tyrosine on the C-terminal tail, or

displacement of the SH2 domain, are not required for Nef-mediated HCK activation, suggesting

this method of SFK activation of an otherwise inactive kinase is unique to Nef [164]. This Nef-

HCK interaction has spurred the structure-based design of novel HCK inhibitors that can reduce

HIV-1 infection without causing cell cytotoxicity [246, 247]. In transgenic mice expressing

mutant Nef lacking the PxxP motif, none of the mice developed an AIDS-like disease [168].

However, mice expressing wild type Nef and HCK knockout only showed delayed onset of the

AIDS-like phenotype [168]. This suggests the SH3 binding ability of Nef is critical for

developing severe AIDS-like disease, but that it depends on the interaction with a more essential

factor that binds PxxP on Nef, perhaps another SFK such as c-SRC.

Nef-mediated activation of HCK has multiple affects on intracellular HIV-1 replication, surface

receptor expression levels and cell motility. Similar to Nef-LCK interactions, HIV-1 Nef

inhibits the anterograde transport of HCK to plasma membrane microdomains, causing HCK to

accumulate at recycling endosomes and the trans-Golgi network [167]. Through HCK, Nef

31

perturbs the intracellular maturation and trafficking of receptors destined to the plasma

membrane, such as CSF-1 receptor (CSF-1R) expression on macrophages [248]. M-CSF is a

cytokine released during viral infection, and promotes the differentiation of macrophages.

However during HIV-1 infection, Nef activation of HCK in the trans-Golgi network

donwregulates surface expression of mature CSF-1R by causing accumulation of under-N-

glycosylated CSF-1R [248]. It was determined that Nef-activated HCK disrupts the distribution

of Golgi Matrix protein 130 (GM130), which is required for efficient protein glycosylation

[249]. Moreover, Nef-activated HCK induced ERK signaling that caused serine phosphorylation

of the GM130-interacting protein Golgi Reassembly-Stacking Protein 1 (GORASP1), causing

this structural protein to unstack Golgi cisternal membranes [249]. The Nef interaction with the

SH3 domain of HCK is also responsible for signalling that downregulates MHC I surface

expression, potentially enabling infected cells to evade killing by CD8+ cytotoxic T-cells [165].

In immature dendritic cells, HIV-1 Nef also induces the downregulation of CD1 surface

receptors, a class of non-MHC lipid antigen-presenting proteins [250]. This effect was mediated

by activated HCK and p21-activated kinase 2 (PAK2) [250]. The PxxP-SH3 interaction between

Nef and HCK can also enhance the incorporation of HCK into budding viral particles from 293T

cells [251]. In addition, Nef inhibits the amoeboid migration pattern of HIV-1 infected

monocytes-derived macrophages, promoting instead mesenchymal migration in vitro [252].

Mesenchymal motility requires podosome regulation and extracellular proteolysis of the matrix,

and it was found that Nef alters the stability, size and proteolytic function of podosomes through

increased HCK activity and Wiskott-Aldrich Syndrome protein (WASp) [252]. This Nef-HCK-

mediated migration reprogramming in macrophages could have important effects on the

dissemination and spread of HIV-1 infection in vivo, as transgenic mice expressing Nef show

increased tissue infiltration [252] and HIV-1 patient tissues accumulate macrophages during

infection [253].

While Nef-induced activation of HCK facilitates HIV-1 infection, HCK kinase activity impairs

later stages of virus assembly and release. This HCK activity is counteracted by viral Vif. Using

living cell fluorescence microscopy, it has been observed that Vif multimerization, an indication

of its role in Gag viral assembly and genomic RNA packaging into viral particles, is altered by

32

HCK expression in HeLa cells [254]. In HCK-expressing cells infected with Vif-deleted HIV-1,

both the release of viral particles and subsequent virion infectivity are strongly reduced [255].

Similar to FYN, Vif has been shown to bind the SH3 domain of HCK, reducing its activity and

preventing autophosphorylation of HCK tyrosine kinase [255]. Also like FYN, HCK

phosphorylates tyrosine on APOBEC3G, increasing the levels of this restriction factor being

packaged into budding virions [238]. As expected, HIV-1 expressing Vif can mitigate this by

reducing HCK activity [238]. Thus, the opposing effects of Nef and Vif on HCK activity

demonstrate the complexity of HIV-1 fine-tuning SFK activity, both in their location and timing,

during the virus replication cycle.

LYN

LCK/c-YES novel tyrosine kinase (LYN) is primarily expressed in hematopoietic cells, neural

cells, and liver tissue [256-258]. LYN kinase activity has important function in regulating

degranulation of mast cells, erythrocyte differentiation, and cell activation [259]. In particular,

LYN has been implicated downstream of B-Cell Receptor (BCR) engagement [260]. Activation

of the BCR receptor leads to LYN phosphorylating Immunoreceptor Tyrosine-based Activation

Motifs (ITAMs) on the cytosolic portions of receptor proteins, recruiting and activating Spleen-

associated tyrosine Kinase (SYK), Phospholipase C gamma 1 (PLC-γ1) and Phosphoinositide 3-

Kinase (PI3K), which further transduce activation signals, induce Ca2+

mobilization, and

promote B-cell proliferation and cell differentiation [194, 261, 262].

As with LCK, HCK and c-SRC, LYN has been shown to bind the Nef PxxP motif through its

SH3 domain, causing allosteric displacement of the SH2-linker, which increases LYN kinase

activity [164]. LYN and HCK share a key Ile residue in their SH3 domain, responsible for their

high affinity binding to a hydrophobic pocket within the core of Nef [164]. However, a

biological role for Nef activation of LYN during HIV-1 infection has yet to be described. Yet

the interactions between LYN, HCK or c-SRC with Nef are highly conserved, as shown with

different Nef proteins isolated from HIV-1 M-group subtypes [263]. In these experiments

testing infection of CEM-T4 lymphoblast cells with chimeric HIV-1 expressing each Nef

protein, Nef-SFK binding could be uniformly disrupted with the compound DFP [263]. This

33

demonstrates that Nef-dependent activation of SFKs are a conserved feature of M-group HIV-1

isolates.

Studies have also demonstrated other roles of LYN kinase activity during HIV-1 infection, with

particular focus on alterations to hepatocytes and dendritic cells. Coinfection with HCV and

HIV-1 has led to research into the effects of HIV-1 envelope proteins on hepatocytes, as patients

with both viruses are more likely to develop liver disease or liver cirrhosis [257]. HIV-1 gp120

has been found to cause bystander apoptosis of uninfected hepatocytes through a Signal

Transducer and Activator of Transcription 1 (STAT1) signaling pathway that involved activation

of LYN, mitogen-activated protein kinase 14 (p38α), and Protein Kinase C (PKC) [257]. In

immature monocyte-derived DCs, it has been demonstrated that inhibiting LYN with the SFK

inhibitor PP2, or reducing LYN expression with targeted siRNA, leads to greater HIV-1

production when co-cultured with primary CD4+ T-cells [264]. This finding suggests LYN may

play an important role in the transfer of HIV-1 from DCs, as LYN has previously been described

to associate with the cytoplasmic tail of DC-SIGN [265]. Furthermore, LYN kinase activity has

been implicated in immune evasion strategies of HIV-1 in immature DCs as they respond to free

HIV-1 virions and complement-opsonized HIV-1 [266]. While exposure of immature DCs to

free virus induced proinflammatory cytokines IL-1β, IL-6, and Tumor Necrosis Factor alpha

(TNF-α), these were dampened in cells treated with complement-opsonized HIV-1 [266].

Instead, these immature DCs showed a different signaling pattern that involved the activation of

IFN regulatory factor 3 (IRF3) and phosphorylated LYN, leading to enhanced infection of the

immature DCs [266]. It is hypothesized that C3-opsonized HIV-1 could be signaling through

TLRs and complement receptor 3 (CR3), which leads to a LYN/PI3K pathway that suppresses

inflammatory responses in immature DCs [266]. These findings suggest HIV-1 could be

subverting the host complement system in a LYN-dependent manner, which may have

implications in immature DCs of the genital tract and rectal mucosa, as these are one of the first

cell types to contact HIV-1 shortly after sexual transmission of the virus.

The effects of LYN kinase activity during HIV-1 infection have been most documented in

monocytes and macrophages, downstream of CCR5 and CXCR4 receptor stimulation. Treating

monocytes-derived macrophages with Macrophage Inflammatory Protein 1 Beta (MIP-1β) leads

34

to activating a CCR5/LYN/ERK-1/2 pathway, which can similarly be triggered by stimulation

with HIV-1 gp120 [267]. This inappropriate macrophage activation by gp120 can induce the

production of TNF-α and other proinflammatory cytokines, which could contribute to AIDS

pathologies and HIV-1 associated dementia [267]. A separate study on primary human,

monocytes-derived macrophages, also found that stimulation of CCR5 with gp120 or whole

virions triggers IL-1β release, which depended on coupling of the Gαi subunit to the CCR5

receptor [268]. This gp120/CCR5 stimulation also led to concomitant PTK2B and PI3K

activation, translocation of both of these signaling proteins from the cytoplasm to the plasma

membrane, and formation of a signaling complex with activated LYN tyrosine kinase [268].

Monocytes migration is also regulated by LYN activity downstream of CXCR4 receptor

engagement with its ligand, SDF-1α [269]. When Brain Microvascular Endothelial Cells

(BMVEC) are activated with TNF- α, IL-1β or treated with HIV-1 gp120, it has been observed

that SDF-1α-treated monocytes no longer adhere to Intercellular Adhesion Molecule 1 (ICAM-1)

ligands, but instead become migratory [269]. Conversly, targeted siRNA knockdown of LYN

prevented the SDF-1α-mediated migration of monocytes, promoting monocytes attachment to

ICAM-1 on activated BMVECs [269]. This data strongly suggests that LYN kinase regulation

of SDF-1α-activated monocytes migration could have an important role in the infiltration of

monocytes into the CNS and past the blood-brain barrier, which may contribute to the various

neurological disorders associated with chronic HIV-1 infection.

FGR, c-YES and BLK

For the final three SFK members, Feline Gardner-Rasheed tyrosine kinase (FGR), cellular

homolog of the Yamaguchi sarcoma virus oncogene (c-YES) and B lymphocyte kinase (BLK),

less is known about their roles during HIV-1 infection.

Expressed exclusively in hematopoietic cells, FGR is found in myeloid cells and B-cells, and

localizes to plasma membrane ruffles [195]. It acts a negative regulator of cell migration and

adhesion downstream of integrin beta-2 (ITGB2) signaling [270]. FGR overexpression has been

associated with a subset of myeloid leukemia and ovarian tumors [271, 272]. Interestingly, the

amino-acid sequence of FGR shares a limited but significant N-terminal sequence homology

35

with HIV-1 Nef [273]. Despite strong expression in macrophages and overlap in signaling

functions with HCK, FGR does not bind or become activated by Nef, likely because FGR lacks

the critical Ile residue in its SH3 domain [164]. In a study of the effects of HIV-1 gp120 on

downstream signaling in naïve B-cells, it was determined that gp120 binds integrin α4β7,

restricting proliferation [244]. This signaling was associated with increased production of the

immunosuppressive cytokine Transforming Growth Factor Beta 1 (TGF-β1), increased

expression of the inhibitory surface receptor FcRL4, and downregulation of FGR expression

[244]. Thus, HIV-1 may be subverting early HIV-1-specific humoral immune responses by

causing B-cell dysfunction through integrin signaling through FGR. Early HIV-1 infection also

appears to alter FGR expression in vaginal epithelial cells. Compared with untreated control

cells, vaginal epithelial cells treated with HIV-1 gp120 showed upregulation of FGR kinase

expression, which occurred in concert with genes that promote inflammation and proteases that

are capable of weakening the vaginal epithelium [274]. Accordingly, FGR may have unique

signaling functions that are tissue specific during HIV-1 infection, reminiscent of how LCK and

HCK exhibit up- and down-regulated activity at specific stages of the HIV-1 replication cycle.

In contrast with FGR, c-YES tyrosine kinase is ubiquitously expressed in many cells and tissues,

having an essential role in regulating cell growth, cell survival, cell adhesion, cytoskeletal

rearrangements, and cell differentiation [275-277] . c-YES signaling has important functions in

glucose activation of the cell cycle, translocation of Epidermal Growth Factor Receptor (EGFR)

into the nucleus during tumor progression, and Platelet-Derived Growth Factor Receptor (PDGF-

R) induced chemotaxis [278-280]. c-YES also has an important role in mediating chemokine-

directed T-cell motility [281]. Jurkat T-cells treated with SDF-1α stimulates CXCR4, which

induces c-YES to phosphorylate Collapsin Response Mediator Protein 2 (CRMP2) at Tyr479,

leading to T-cell polarization and lymphocyte migration [281]. As with FGR, the SH3 domain

of c-YES does not permit interactions with HIV-1 Nef [164]. In a study of HIV-1 Tat modifying

global gene expression in primary CD4+

T-cells, it was found after 24 hrs of Tat exposure that c-

YES showed a modest, yet significant reduction in gene expression [282]. This correlated with

the CD4+ T-cells increasing secretion of 11 different cytokines, with notable production of IL-

17, and proinflammatory gene expression reminiscent of the Th17 phenotype [282]. However,

36

this finding is incongruent with another study examining the role of c-YES and apoptosis-related

genes during HIV-1 infection of Jurkat T-cells [283]. In this work, Jurkat T-cells infected with

HIV-1 showed increased c-YES expression within 3 days, as confirmed by Western blot [283].

The authors suggest that c-YES may be signaling downstream of the Fas receptor (FasR), which

can oligomerize and trigger apoptosis through the Death-Inducing Signaling Complex (DISC)

[283].

Lastly, BLK is a tyrosine kinase expressed in pancreatic β cells and hematopoietic cells, with

essential function in B-cells in particular [284, 285]. It participates in pre-BCR signaling, B-cell

development, and B-cell differentiation [286, 287]. As with other SFKs, BLK transmits signals

from cell surface receptors, and has been implicated in B-cell associated autoimmune disorders

[288]. Little is known of changes to BLK activity in response to HIV-1 infection, although a

BLK gene variant has been found to disrupt B-cell proliferation and the ability to elicit antigen-

specific CD4+

T-cells in patients suffering from Common Variable Immunodeficiency (CVID)

[196]. A genome-wide study of host proteins involved in early HIV-1 infection found that

siRNA knockdown of BLK could reduce infection of VSV-g pseudoenveloped HIV-1 in 293T

cells [289]. However, confirmatory experiments exposing primary B-cells to extracellular HIV-

1 proteins such as gp120, Nef or Tat, have yet to ascribe a function for BLK during HIV-1

infection.

37

1.2.5 Role of c-SRC in HIV-1 Infection

c-SRC is the canonical member of the SFKs, often expressed as a cytosolic proto-oncogene

tyrosine kinase. In humans, it is expressed in nearly all cells and tissues, with high levels of

protein found in the brain (neuronal splice variant), testis, platelets and PBMCs [190, 290-292].

c-SRC is expressed in CD4+ T-cells 12 hours following TCR-CD3 activation [293]. Its primary

functions are thought to play a role in fetal organogenesis, differentiation, cell cycle progression,

proliferation, cell survival, cell adhesion, and cell migration via cytoskeletal rearrangement at

adhesion networks [294]. c-SRC is a key signaling molecule that integrates multiple signal

inputs that induce a variety of downstream signal cascades, as evidenced by the 132 c-SRC

mediated phosphorylation sites in the human proteome, 64 known substrates, and over 204

proteins that interact with c-SRC [294]. c-SRC is predominantly repressed in an inactive state,

and transiently activated during cellular processes such as mitosis [189]. Conversely, the kinase

is constitutively active in abnormal states associated with human cancers and viral infection of

chickens by RSV [295, 296]. c-SRC is often associated with cellular membranes, in particular

the plasma membrane, endosomal membranes and the nuclear envelope [297, 298]. The defined

subcellular locations of c-SRC are important for the regulation of specific cellular events, such as

cytoskeletal rearrangement, membrane trafficking and cell cycle progression [189].

Given the essential role of c-SRC in cellular processes, and its potential to cause aberrant tumor

growth or differentiation when constitutively active (ex. colon, breast or prostate cancers [296,

299, 300]), it is unsurprising that the cell regulates c-SRC activation with multiple post-

translational modifications (see Figure 1.7 for c-SRC protein structure and regulation, originally

published in [199]). Phosphorylation of the C-terminal tyrosine residue Tyr530 leads to

intermolecular folding and binding to the SH2 domain, suppressing c-SRC tyrosine kinase

activity [301]. Interestingly, the regulatory Tyr530 is absent in the truncated v-SRC protein,

accounting for its constitutive activity [302]. The conformational change caused by Tyr530

phosphorylation also allows intramolecular binding of the SH3 domain to a proline-rich motif,

further suppressing c-SRC activity [303]. A variety of c-SRC binding proteins regulate c-SRC

activity by disrupting its SH2 and SH3 intramolecular interactions. For example, PDGFR and

38

Fig. 1.7: c-SRC domains, tertiary structure, and regulation. (A) The seven domains

characteristic of the SFKs are shown for c-SRC. CSK and CHK phosphorylation of Tyr530 can

lock c-SRC in an inactive state [199]. (B) Crystal structure of activated c-SRC. The protein

domains are the same colours as in (A) [199]. (C) Schematic of activated myrstilated c-SRC at

the plasma membrane. (D) Inactive state of c-SRC.

39

Focal Adhesion Kinase (FAK) can bind the c-SRC SH2 domain and activate the enzyme [183,

304], while Sin is a protein that can bind the SH3 domain to activate c-SRC [305]. Moreover,

dephosphorylation of Tyr530 by cellular Protein Tyrosine Phosphatases (PTPs) relieves this

intramolecular inhibition of c-SRC kinase activity [306]. Full activation of the enzyme requires

a second major tyrosine residue, Tyr419, located in an activation loop in the catalytic domain

[302]. Phosphorylation of c-SRC at Tyr 419 displaces this residue from the substrate binding

pocket, permitting the kinase to phosphorylate substrate targets. Tyr419 can be phosphorylated

by other tyrosine kinases or through autophosphorylation [307]. Therefore, phosphorylation of

Tyr419 acts as a positive regulator of c-SRC activity, while phosphorylation of Tyr530 acts as a

negative regulator to suppress c-SRC activity [302].

c-SRC has multiple phosphatase and kinase binding partners that tightly control its kinase

activity. Phosphatases that dephosphorylate c-SRC Tyr530 to activate the enzyme include:

Receptor-type tyrosine-protein phosphatase alpha (PTPRA, PTP-α) and PTP non-receptor type 6

(PTPN6 or SHP-1), 11 (PTPN11 or SHP-2) and 12 (PTPN12 or PTP-PEST) [306, 308-310]. C-

terminal SRC Kinase (CSK) and C-terminal SRC kinase-Homologous Kinase (CHK) are two

kinases that counteract these effects at Tyr530 [191, 311]. In resting T-cells, CSK binding

protein (Cbp) and phosphoprotein associated with glycosphingolipid-enriched microdomains

(PAG) are constitutively phosphorylated, recruiting CSK which prevents the activation of c-SRC

[312]. In contrast, T-cell activation induces rapid dephosphorylation of Cbp/PAG, reducing

PAG-mediated inhibition of c-SRC by CSK [312].

As previously mentioned, the subcellular location of c-SRC has important function in the

signaling cascades that it regulates. Myristoylated c-SRC associated with the plasma membrane

regulates cell growth and cell proliferation downstream of growth factor receptors [304]. Plasma

membrane c-SRC also localizes at focal adhesion plaques and adheren junctions between cells

[313], which is dependent on an intact actin cytoskeleton [314]. c-SRC is also localized to the

perinuclear Golgi region of the cell, accounting for 30-40% of the total c-SRC cellular protein

[315]. Within the nucleus, c-SRC has been proposed to regulate cell cycle progression and entry

into mitosis by interacting with proteins that regulate cell division [298]. It has been shown that

the SH3 domain of c-SRC interacts with Sam68, a nuclear protein with 6 proline-rich regions

40

and a tyrosine-rich C-terminal domain [316]. Sam68 is a multifunctional RNA-binding protein

that is required for transactivation of Rev function and the cytoplasmic translation of HIV-1

RNA into proteins [317]. Cytoplasmic mutants of Sam68 have been shown to selectively inhibit

the translation of HIV-1 RNA transcripts [317]. This suggests Sam68 directly participates in the

nuclear exportation of unspliced and singly-spliced HIV-1 RNA. Moreover, overexpression of

Sam68 can enhance the expression of viral p24, through translational regulation of HIV-1 RNA

[317]. Beyond interactions with Sam68, c-SRC has also been found within the nucleolus [298].

Despite all this localization knowledge, little is known of the cytosolic effects of c-SRC signal

transduction away from the plasma membrane, or the specific substrates and binding proteins

that interact with cytosolic c-SRC. Moreover, the potential role of cytosolic c-SRC during the

lifecycle of HIV-1 infection has not been established or well defined.

Previous work has demonstrated that the Jurkat T-cell line exhibits robust c-SRC activity after 30

minutes of HIV-1IIIB infection [214]. Moreover, treatment of activated PBMCs from healthy

human donors with a synthetic peptide expressing the CD4-binding region of gp120 (Peptide T),

could induces an 11-fold increase in c-SRC kinase activity within 15 minutes [318]. As with

other SFK members, viral Nef binds to c-SRC through allosteric displacement of SH3 domain

interactions, requiring the PXXPXR motif on c-SRC [164]. Yet the direct effect of Nef on c-

SRC activity in vivo during infection is not clear [164, 319]. Relative to HCK, c-SRC lacks the

SH3 domain Ile residue that promotes high Nef binding affinity [164]. This creates an SH3

domain more constrained by hydrogen bonds, and may take a conformation less compatible for

Nef interactions [164]. In this study, Nef binding to c-SRC alone could not induce strong c-SRC

kinase activity in vitro [164]. Yet in another paper on immortalized podocytes, Nef was shown

to increase c-SRC activity and induce proliferation in this cell line by inducing activation of

STAT3, the Ras family of small GTPases (Ras) and MAPK1[320]. These Nef-c-SRC

interactions underpinning aberrant glomerular podocyte growth may contribute to HIV-1

associated nephropathy, which can lead to chronic renal failure in patients living with HIV-1

[320].

Kinase inhibitor experiments have also suggested a potential role for c-SRC during HIV-1

infection. A study examining the transfer of HIV-1 from primary DCs to CD4+ T-cells

41

demonstrated that CD4+ T-cells pre-treated with the broad SFK inhibitor PP2, reduced the p24

produced after 6 days of HIV-1JR-CSF or NL4-3balenv infection [264]. However, it is difficult to

ascertain the direct effects of c-SRC inhibition with pyrazolopyrimidines, such as PP1 or PP2, as

they inhibit LCK and FYN at similar IC50 values as c-SRC [184]. PP1 and PP2 also have many

off targets, such as CSK, which can counteract the effect of either drug on SFK activity in cells

[184]. Research into the effects of LCK, FYN and c-SRC in the budding of HIV-1 virus-like

particles suggests that LCK and FYN both facilitate this step of the viral lifecycle, where as c-

SRC had no effect on virus released from transfected 293T cells [227]. Thus c-SRC likely

affects HIV-1 replication at an earlier stage of the viral lifecycle. Another study investigating the

permeability of endothelial cells in the dissemination of HIV-1 found that pre-treating Lung

Lymphatic Endothelial Cells (L-LECs) with SRC inhibitor 1 (SRC-I1) induced

hyperpermeability of the lymphatic endothelial barrier [321]. Moreover HIV-1 gp120 could

induce similar L-LEC hyperpermeability that required SFK modulation of the cytoskeleton

[321]. However, the direct effects of c-SRC during the early entry events of HIV-1 in CD4+ T-

cells, and whether they modify the host cytoskeleton, has not yet been determined.

c-SRC involvement in cytoskeletal organization and membrane trafficking via its binding

partner, protein tyrosine kinase 2 beta (PTK2B), is a potential signaling pathway exploited by

HIV-1 during early infection [147, 197, 322]. PTK2B/c-SRC activity has also been implicated

in the migration and podosome formation of immature DCs exposed to soluble gp120, which

may enhance the mucosal transmission of HIV-1 as these cells migrate in response to HIV-1

envelope protein [323]. Immature DCs treated with gp120 enhanced the phosphorylation of c-

SRC and PTK2B, which activated Rac-1 subfamily of Rho small GTPases (Rac-1) signaling and

subsequent paxillin phosphorylation, an essential adhesion molecule that facilitates podosome

ring structure [323]. Whether PTK2B/c-SRC signalling plays a similar role in the migration of

CD4+ T-cells during HIV-1 infection, has yet to be evaluated.

42

1.2.6 Role of PTK2B in HIV-1 Infection

PTK2B, the second member of the FAK family, is a 112 kDa non-receptor tyrosine kinase highly

expressed in hematopoietic, neuronal and epithelial cell lineages [324-326]. Slightly smaller

than FAK, the two proteins share 45% sequence identity [327] (see Figure 1.8 for PTK2B

structure, originally published in [328] and [329]). An alternatively spliced form of PTK2B is

expressed in the spleen, with a 42 amino acid deletion within the C-terminal domain [324].

PTK2B contains tyrosine phosphorylation sites that recruit binding of SH2 domains of proteins

such as Growth Factor Receptor-Bound protein 2 (GRB2) and c-SRC [197]. In addition, the C-

terminal end of PTK2B contains two conserved proline-rich motifs that can further bind proteins

containing an SH3 domain [330]. The C-terminus also contains a Focal Adhesion Targeting

(FAT) sequence that allows for binding to paxillin or leupaxin [331]. Unique to the FAK family,

PTK2B has a central tyrosine kinase domain flanked by an N-terminal Four point

1/Ezrin/Radixin/Moesin (FERM) domain, which regulates PTK2B complex formation and

phosphorylation [332]. Like c-SRC, multiple phosphorylation sites tightly regulate PTK2B

function. Located in the linker region between the FERM domain and kinase domain is Tyr402,

a residue critical to the activation of PTK2B and the recruitment of SFKs [197]. The activation

loop in the kinase domain also contains Tyr579 and Tyr580, two amino acids that when

phosphorylated, induce maximal PTK2B kinase activity [333].

Various cell models have explored the interaction between PTK2B and c-SRC, which link Gi-

and Gq-coupled receptors with downstream effects within the cell [197]. C-terminal

dephosphorylation of c-SRC at Tyr530 by PTPs is one of the earliest steps to c-SRC activation

[306, 308-310]. The SH2 domain of c-SRC then becomes free to bind ligands such as PTK2B, at

the PTK2B autophosphorylation site Tyr402 [197]. The binding of c-SRC to PTK2B regulates

both the activation of c-SRC kinase activity and its cellular localization, bringing c-SRC in close

proximity of potential substrates. For instance, autophosphorylation of PTK2B creates a binding

site for the c-SRC SH2 domain, localizing c-SRC to focal adhesions where it can phosphorylate

paxillin to regulate cell migration [183].

43

Fig. 1.8: PTK2B domains, tertiary structure, and regulation. (A) PTK2B protein domains.

The c-terminus FERM domain negatively regulates catalytic activity. The FERM domain is

followed by a proline-rich region (PRR), and Tyr402 that when phosphorylated, can bind the

SH2 domain of c-SRC. Next is the kinase domain with two positive regulatory tyrosines

(Tyr579 and Tyr580), and two more PRRs. The N-terminus ends with a focal adhesion targeting

(FAT) domain that can bind paxillin at focal adhesions [328]. (B) Crystal structure of the

PTK2B kinase (cyan) and FERM (green) domains. The three lobes (F1-F3) of the FERM

domain are shown, as well as Ca2+/

calmodulin binding motifs (orange) [329]. (C) Schematic of

activated PTK2B downstream of voltage-gated Ca+2

ion channels.

44

PTK2B is most well studied for its participation in integrin receptor signaling pathways at focal

adhesions in adherent cell types [334], signaling during apoptosis [335], cell migration [326], as

well as responding to elevated calcium signals [336]. Extracellular matrix proteins binding to

integrins are important for transducing signals that promote cell growth, cell survival, and cell

migration. In this integrin pathway, PTK2B colocalizes with integrin receptors at cell contact

sites, termed focal adhesions, to phosphorylate structural proteins such as paxillin, which directly

associate with the actin cytoskeleton [334]. PTK2B also recruits SFKs and other focal adhesion

proteins to activate signaling targets such as Rac1, a member of the Rho Guanosine

Triphosphatase (GTPase) family [337]. In the absence of integrin signaling that promotes

adhesion-dependent cell survival, PTK2B activation can induce early apoptotic signaling by

activating caspase-3, SFKs, and increase DNA fragmentation [335]. In conjunction with integrin

receptors, PTK2B transduces migratory signals downstream of growth factor, antigen,

chemokine and cytokine receptors [147, 326, 338]. PTK2B is increasingly being recognized for

its role in responding to elevated intracellular calcium signals as a Calcium-Dependent Tyrosine

Kinase (CADTK). An influx of extracellular calcium via voltage-gated calcium channels or

cytosolic release from intracellular stores can induce an increase in PTK2B tyrosine kinase

activity [339]. It has also been suggested that calpains, Ca2+

-dependent cytosolic cysteine

proteases, cleave PTK2B in order to regulate cell attachment and motility through assembly and

disassembly at focal adhesions [336].

Within T-cells, PTK2B has many important roles in T-cell stimulation and effector cell functions

[340, 341]. For instance, PTK2B is essential for LFA-1 dependent CD8+ T-cell activation,

migration and cell adhesion [341], forming micro-adhesion rings around the TCR to stabilize the

immunological synapse [342], establishing cell polarity [180], and donwregulating activated

surface receptors. In helper CD4+ and cytotoxic CD8

+ T-cells, TCR and integrin stimulation

lead to tyrosine phosphorylation of PTK2B, enhancing its catalytic activity, and causing PTK2B

translocation to sites of cell contact [343]. PTK2B also participates in downstream signaling of

TCR and CD28 costimulation that promote IL-2 production [344]. Unique in T-cells, PTK2B

exhibits a biphasic burst in activity shortly after TCR engagement that is dependent on SFK

activation [340]. Both Tyr402 and Tyr580 on PTK2B are phosphorylated early (5 min) and then

45

late (30-60 min) after TCR activation, causing two distinct phases of paxillin phosphorylation

and actin polymerization [340]. It has been hypothesized that the two periods of PTK2B

activation control the cytoskeletal assembly and then disassembly necessary for optimal T-

cell/APC contact, which occurs for approximately 1 hr [340]. In the first burst, PTK2B

facilitates cytoskeletal changes that may reinforce the T-cell and APC contact, while the second

burst of PTK2B activity disassembles the actin cytoskeleton, and may help disengage the T-cell

from the APC.

Past research suggests PTK2B signaling may influence HIV-1 binding and entry of CD4+ T-cells

through signaling downstream of G protein-coupled receptors engaged during viral co-receptor

binding [147], and responding to ATP that enhances viral membrane fusion to the target T-cell

[345]. After natural stimulation of surface CCR5 with Chemokine (C-C motif) Ligand 5 (CCL5)

or MIP-1β, or CXCR4 stimulation with SDF-1α, PTK2B becomes rapidly phosphorylated [147].

This suggests that HIV-1 binding to either surface receptor might mimic natural ligand

engagement and produce physiologically similar signals. Indeed, infecting the T-cell line DU6

(CCR5+) with JR-FL (R5) pseudoenveloped virus induced PTK2B activation, as did mixing

HL60 cells (CXCR4+) with 293T cells expressing HXB2 (X4) envelope protein [147]. Yet

whether PTK2B activation is simply a byproduct of HIV-1 envelope engagement, or has a

functional role in the intracellular lifecycle of HIV-1 post-fusion, has yet to be determined.

Binding of HIV-1 gp120 to CD4 and a chemokine receptor also induces mechanical membrane

stress, stimulating the release of ATP through pannexin-1 hemichannels [345]. In the

extracellular space, ATP then acts as an autocrine and paracrine signal, activating the purinergic

receptor P2Y2 on the cell surface, which are coupled to G-proteins that recruit PTK2B that

subsequently autophosphrylates at Tyr402 to become activated [345]. P2Y2 also has an SH3

binding domain, which can directly recruit PTK2B and induce its phosphorylation and c-SRC

activation [182]. These provide even more avenues by which PTK2B recruits signaling

complexes that contain SFKs or actin interacting proteins that may enhance HIV-1 fusion upon

entry.

Once HIV-1 enters a T-cell, studies suggest FAK apoptotic signaling is blocked [346], and that

PTK2B promotes cell migration during HIV-1 infection [323]. After gp120 engages CD4 and

46

CCR5, FAK localizes to the inner face of the plasma membrane at these receptors [346]. It then

becomes phosphorylated and cleaved by caspase-3 and caspase-6, promoting apoptosis and

inactivating FAK in uninfected cells [347, 348]. Interestingly, the interaction of the FAT domain

of FAK with the cytosolic portion of CD4 shares similarity with the interaction of HIV-1 Nef

with CD4 [346]. Both FAK and Nef bind CD4 in the same region and with similar affinity

[349], yet cause opposing signalling effects. The FAK-CD4 interaction with gp120 can trigger

apoptosis of a bystander T-cell, through FAK cleavage by caspases [348]. However, the Nef-

CD4 interaction can block this pro-apoptotic signal, permitting an infected T-cell to survive and

productively produce virions [346]. Whether PTK2B has a similar role as FAK, to induce

apoptosis signaling that is blocked by Nef in CD4+

T-cells during HIV-1 infection, has yet to be

assessed. In addition, the potential effect of PTK2B activation on CD4+ T-cell migration during

HIV-1 infection has not been determined. As mentioned earlier, gp120-induced transendothelial

migration of immature DCs relies on c-SRC signalling, as well as downstream signaling by

PTK2B, paxillin, and Rac1 [323]. In these cells, PTK2B activates the Rho GTPase Activated

CDC42 Kinase 1 (ACK1), promoting actin polymerization, ring formation, and nucleation of

actin filaments within the podosome core [323]. Taken together, there are multiple mechanisms

by which c-SRC and PTK2B signaling could be cooperating (or acting independently) to alter

intracellular HIV-1 infection post-entry in CD4+ T-cells.

47

1.3 Ebola Virus (EBOV)

1.3.1 ZEBOV Epidemiology, Transmission and Replication Cycle

In 1976, WHO medical staff and USA Center of Disease Control (CDC) researchers witnessed

two separate outbreaks of an unknown contagious disease in Nzara, southern Sudan (284 cases,

151 deaths), and in Yambuku, northern Zaire (318 cases, 280 deaths) [350, 351]. The disease

caused fever, joint and muscle pain, rash, abdominal pain, diarrhea with blood, and rapid

progression to death, leading to regional panic in both locations. A new virus was isolated by

Dr. Peter Piot in Belgium and further characterized by a CDC researcher, Dr. Karl Johnson, from

the blood sample of an infected Belgium nun in Zaire [352]. The virus was named after the Ebola

River located near the original Zaire outbreak, in what is now the Democratic Republic of the

Congo [352]. It was later discovered that both outbreaks were caused by two distinct species of

ebolavirus, now classified as Sudan ebolavirus (SUDV) and Zaire ebolavirus (ZEBOV) [353].

Three more have been isolated in the Ebolavirus genus: Bundibugyo ebolavirus (BDBV),

discovered in Uganda [354], Reston ebolavirus (RESTV) from the Philippines that infects

cynomolgus macaques and is non-pathogenic to humans [355], and Taï Forest ebolavirus

(TAFV), isolated from a chimpanzee in Côte d’Ivoire [356]. From 1976 to 2013, there have been

24 separate Ebola outbreaks primarily clustered in central Africa, with mortality rates ranging

between 25-90% [6, 357]. The difference in host fatality and disease severity attributed to each

ebolavirus strain is still under investigation [357]. None of these outbreaks compare with the

unprecedented 2014-16 ZEBOV epidemic in West Africa that led to 28,616 cases and 11,310

deaths in Liberia, Guinea and Sierra Leone (see Figure 1.9, originally published in [5]). This

spurred significant developments in Ebola virus therapeutics and vaccines that were tested for

safety and efficacy in human trials during the outbreak. While a licensed treatment has proven

elusive [25], a successful vaccine candidate has recently been announced [26]. Significant

challenges were faced with implementing clinical trials in resource limited settings, including

global support and preparedness, maintaining public trust, ethical concerns of implementing

randomized control trials during an outbreak, and a decline in Ebola cases once clinical trials

were initiated [25].

48

Fig. 1.9: Spread of ZEBOV in West Africa during 2014-16. The index case was a small boy

in Guinea from the Guéckédou prefecture, who died in December 2013 (yellow star). EVD then

spread quickly from his family to neighboring villages. By May 2014 the virus had spread to

Conakry, the capital city of Guinea, and cases were being reported in Monrovia, the capital city

of Liberia, by mid June. The outbreak quickly spread to Sierra Leone and intensified in all three

countries from July to October in 2014. Smaller outbreaks in Nigeria and Mali were contained.

The epidemic began to wane in November 2014 from international control efforts. All known

chains of transmission ended by June 2016, a year and a half since the first case in the region [5].

49

The West Africa ZEBOV outbreak began with cases reported in Guinea during December of

2013, then spread quickly to neighboring countries Liberia and Sierra Leone [358]. From a

single introduction into the human population, it caused an overall fatality rate of 40% over

several months [5]. The variant responsible for the epidemic was isolated and determined to be

genetically related to ZEBOV, classified as the EBOV-Makona reference strain [359]. In August

2014 an unrelated outbreak of ZEBOV, caused by the variant EBOV- Lomela, occurred in the

Democratic Republic of the Congo [359]. Transmission of this variant lead to a shorter ZEBOV

epidemic, with fewer fatalities (66 cases, 49 deaths) [359]. It was not until March 29, 2016 that

the WHO declared an end to the Public Health Emergency of International Concern regarding

the ZEBOV outbreak in West Africa, although sporadic cases continue to occur in Guinea and

Sierra Leone [5, 360]. It is not yet understood why some Ebola survivors are asymptomatic

seroconverters, while others succumb to life threatening conditions collectively called Ebola

Virus Disease (EVD) [361]. Studies of large patient cohorts demonstrate that overall survival

and disease severity could be predicted from initial viral load at time of admission to an Ebola

Treatment Center (ETC) [18, 19]. The unprecedented outbreak also produced the largest group

of Ebola survivors, with ~ 17,000 recovered patients in the three most affected countries, who

exhibit newly described chronic afflictions called post-Ebola disease syndrome [362]. Ebola

survivors are at greater risk for ocular problems (blurred vision, retro-orbital pain), loss of

hearing, difficulty swallowing, abdominal and back pain, fatigue, severe headaches, memory

problems, and confusion, among others [362-364]. After acute infection, ZEBOV has been

found to persist in semen, ocular fluid, cerebrospinal fluid, placenta, and amniotic fluid [365-

367]. A case of a woman contracting ZEBOV in Guinea after sexual intercourse with a male

Ebola survivor 470 days after the onset of his symptoms, who also shed ZEBOV in his semen up

to 531 days after disease onset, demonstrates that infectious virus can be transmitted sexually

from Ebola survivors [360].

Ebola viruses are in the family Filoviridae, and are lipid-enveloped, heavily glycosylated,

filamentous RNA viruses (Figure 1.10, originally published in [368]). The 19-kb genome

consists of a non-segmented, negative-sense single strand of RNA ((-)ssRNA) coding for 7

genes, each separated by short intergenic regions [369]. Flanking both ends of the genome

50

Fig. 1.10: ZEBOV virion and genome structure. (A) The negative sense, single-stranded

RNA genome of ZEBOV is bound to nucleoprotein (NP) minor nuclear protein (VP30), RNA-

dependent RNA polymerase L (L) and polymerase cofactor protein (VP35). Trimeric

glycoprotein (GP1,2) pass through the lipid envelope and facilitate viral entry into cells. Matrix

proteins VP40 and VP24 are located on the cytosolic side of the lipid envelope. Filamentous

ZEBOV virions have variable length, and multiple nucleocapsids are packaged within a single

virus particle. (B) Schematic representation of the 7 gene coding regions of the ZEBOV

genome, flanked by leader and trailer sequences. Variants of GP protein are indicated [368].

51

are extragenic regions called the leader and trailer sequences, which signal for encapsidation and

contain promoters for replication and transcription [370]. The genome encodes for 4 structural

proteins (nucleocapsid protein (NP), glycoprotein (GP1,2), viral protein 24 (VP24) and viral

protein 40 (VP40)), and 3 replication proteins (RNA-dependent RNA polymerase L (RdRP L),

viral protein 35 (VP35) and viral protein 30 (VP30)) [369]. GP1,2 is the only surface

glycoprotein that mediates virus fusion and entry, and the mature trimer of GP1-GP2

heterodimers shares structural similarity with retroviruses such as HIV-1 [371]. Transcriptional

stuttering of the polymerase also produces two soluble GP variants called sGP and small sGP.

In the EBOV cellular replication cycle, a variety of host surface receptors act as cofactors prior

to viral entry (see Figure 1.11, originally published in [368]). These include T-cell

Immunoglobulin and Mucin Domain 1 (TIM-1), integrins, asialoglycoprotein expressed on

hepatocytes, and C-type lectins such as DC-SIGN on dendritic cells and macrophages [372-374].

Upon GP1,2 binding a surface cofactor, the virus enters the early endosome by macropinocytosis

[375]. The endosome vesicle acidifies, and host cysteine cathepsins cleave GP1, which unmask

GP2 [376]. This allows host Niemann-Pick C1 (NPC1) to bind the cleaved GP (GPCL) as the

bonafide entry receptor, triggering a conformational change in GP2 that promotes fusion of the

late endosome and viral membranes, releasing the virus into the cytoplasm [376, 377]. Within 6-

12 hours, the RdRP L forms a complex with NP in the form of cytoplasmic inclusion bodies

outside the nucleus [378]. Polymerase L performs primary transcription of individual mRNAs

from the negative-sense RNA genome, with assistance of VP30 (transcription activator) and

VP35 (polymerase cofactor), and the mRNAs are capped at the 5’ end and polyadenylated at the

3’ tail [379, 380]. Each gene is transcribed sequentially from 3’ to 5’, producing abundant

transcripts of genes at the 3’ end relative to the 5’ end [370]. Thus NP transcripts are the most

abundant, while RdRP L transcripts are the least transcribed. Phosphorylation of VP35 triggers

RdRP L, NP and VP30 to switch to genomic replication, producing full-length positive-sense,

anti-genomic RNA that serve as templates for negative-sense genomic synthesis (secondary

transcription) [381]. Newly translated NP, VP35, VP30 and RdRP L proteins associate with the

progeny negative-sense RNA strands, while GP and sGP mature separately in the endoplasmic

reticulum and Golgi bodies to be

52

Fig. 1.11: The intracellular ZEBOV replication cycle. ZEBOV GP1,2 binds to glycoproteins

such as TIM-1 on the host plasma membrane, triggering macropinocytosis of the virus. Host

cathepsin B and L cleave GP1, which exposes the entry receptor on GP2 that binds host NPC1.

This interaction induces fusion of viral and vesicular membranes in the late endosome. In the

cytosol, viral polymerase L performs primary transcription to produce mRNAs, which are

translated into proteins that collectively form a viral factory (inclusion body) outside the nucleus.

VP35 and VP24 also dampen interferon signaling and RNA-sensing mechanisms, allowing

abundant viral proteins and genomic (-)ssRNA to accumulate at the plasma membrane through

host Endosomal Sorting Complexes Required for Transport (ESCRT) proteins. Nucleocapsid

protein (NP) encapsidating ZEBOV genomic RNA, viral matrix protein VP24, and matrix

protein VP40, together mediate assembly and budding of mature virions from the cell [368].

53

post-transcriptionally modified [378]. Concurrently, membrane-associated matrix proteins VP24

and VP40 are translated and localize to the plasma membrane [382]. When sufficient levels of

NP and negative-sense RNA genomes are produced, VP24 helps assemble viruses at the plasma

membrane, then VP40 induces budding of the newly formed virions [382]. Outside of

replication, VP24 and VP35 have additional roles that help EBOV evade host innate immune

responses, chiefly by antagonizing type I IFN pathways [383].

ZEBOV is transmitted between humans by direct contact with mucosal membranes or bodily

fluids (saliva, sweat, vomit, diarrhea, blood or semen) of an infected or deceased individual

[367]. Following a 6-12 day incubation period, a symptomatic patient will be highly contagious

during the acute phase of EVD. Early symptoms consist of fever, chills, malaise and muscle

pain, which are non-specific and can be confused with other hemorrhagic fevers or viral

infections [357, 367]. When the virus enters broken skin or mucosal surface, antigen-presenting

cells are the preferred initial cellular targets, such as monocytes, macrophages or dendritic cells

[384]. In vitro infection of these cells demonstrates robust expression of inflammatory mediators

IL-1β, IL-6, IL-8, MIP-1α, MIP-1β, MCP-1, and TNF-α [384-386]. Infected cells also release

chemokines that recruit additional monocytes and macrophages to the site of infection, which in

turn become infected [384]. Symptoms at this stage are gastrointestinal (vomiting, diarrhea

abdominal pain, nausea, anorexia), respiratory (nasal discharge, cough, shortness of breath, chest

pain) and vascular (postural hypotension, edema), demonstrating multisystem involvement [387].

As the host immune system attempts to mobilize an adaptive response, ZEBOV may be

impairing the essential role of APCs to stimulate T-cell and B-cell responses required to clear

infection [388]. Moreover, infected APCs secrete soluble GP, which may act as decoys that

counteract neutralizing antibodies [389]. Infection of APCs, coupled with elevated

proinflammatory cytokines and a weak adaptive immune response, are likely why ZEBOV can

disseminate systemically to infect hepatocytes, adrenal cortical cells, and endothelial cells [390].

This results in hepatocellular necrosis that decreases secretion of coagulation proteins, and

abnormal adrenal cortical cells that can no longer maintain blood pressure homeostasis.

In the late stages of illness, symptoms include intravascular volume depletion, hypoperfusion,

electrolyte perturbations and renal impairment [387]. Severe lymphopenia and lymphoid tissue

54

destruction occur, with apoptosis causing the greatest loss of peripheral blood CD4+ and CD8

+ T-

cells [391]. In concert, the large release of TNF-α from infected monocytes and macrophages

induces endothelial permeability, causing vascular leakage and hemorrhage [384]. Together the

blood leakage, extensive cytokine release and viral replication in endothelial cells lead to

hemorrhage syndrome, often characterized by gastrointestinal bleeding [387]. At the terminal

stage of disease, multi-organ failure and shock are the main causes of death.

55

1.3.2 ZEBOV Clinical Trials, Potential Vaccines and New Therapeutics

Despite international efforts to accelerate research into ZEBOV therapeutics and conduct clinical

trials during the 2014-16 West Africa outbreak, there remains no licensed treatment for EVD

[25]. However, there was success with a clinical trial testing a preventative vaccine [26]. A

variety of vaccine platforms have been used to treat EBOV infection in Non-Human Primate

(NHP) models of infection, including: virus-like particle antigen-based vaccines, replication-

deficient DNA and recombinant Adenoviral Vector (rAD) vaccines, and replication-competent

viral vectors that employ recombinant parainfluenza, rabies, Cytomegalovirus (CMV) or

Vesicular Stomatitis Virus (VSV) [392]. However the two most promising vaccine candidates

that reached phase II/III clinical testing during the West Africa ZEBOV outbreak were ChAd3-

EBO-Z and rVSV-ZEBOV-GP [26].

ChAd3-EBO-Z is a non-replicating, recombinant, chimpanzee adenovirus type-3 vector-based

vaccine expressing ZEBOV GP [393]. Earlier studies on cynomolgus macaques demonstrated

that single immunization 5 weeks prior to ZEBOV challenge led to complete protection, but this

dropped to 50% protection when the animals were challenged 10 weeks post-immunization [32].

Pre-existing immunity to ADVs, which also occurs in humans, likely explains the lower than

desired GP-specific humoral response. Thus a booster vaccine with modified vaccinia Ankara

expressing ZEBOV GP (MVA-BN-Filo) was administered 8 weeks after ChAd3-EBO-Z

immunization, leading to complete ZEBOV protection in macaques challenged as late as 10

months post-immunization [32]. In September 2014, the US National Institute of Allergy and

Infectious Diseases (NIAID) and UK Welcome Trust promptly tested the safety, tolerability, and

immunogenicity of ChAd3-EBO-Z in 91 healthy participants in two, single-blind, dose-

escalation phase I trials in Mali and the USA [393]. Another phase I trial was performed on 60

patients in the United Kingdom, and it was found that the vaccine boosted with MVA elicited

strong B-cell and T-cell responses, with no safety concerns reported [394]. The vaccine platform

then entered a phase II/III trial called Partnership for Research on Ebola Vaccines in Liberia

(PREVAIL), which enrolled 27,000 healthy adult participants, and is estimated to complete by

56

June 2020. PREVAIL is a randomized, double-blind, controlled, 3-arm study that is also

evaluating the other most promising vaccine platform, rVSV-ZEBOV-GP .

rVSV-ZEBOV-GP is a live attenuated, recombinant, replication-competent vaccine consisting of

VSV expressing ZEBOV GP [26]. It has the advantage over non-replicating vaccines by

providing longer durability, but possesses the potential risk of reversion to wild type (WT), and

could cause complications in people with compromised immunity [357]. No adverse events

were observed in 80 NHPs immunized with single dose rVSV-ZEBOV-GP [395], or cause

disease in immunocompromised Non-Obese Diabetic (NOD) Severe Combined

Immunodeficiency (SCID) mice [396]. The vaccine could protect 100% of macaques from lethal

ZEBOV challenge up to 6 months after immunization with a single vaccine dose, and confer

50% protection when administered 30 min post-exposure of ZEBOV challenge [33, 397]. rVSV-

ZEBOV-GP safety and immunogenicity in humans was also determined in multiple phase I trials

across Europe and Africa in 2014 [398, 399]. Recent findings from a phase III, cluster-

randomized ring trial in Guinea and Sierra Leone showed this vaccine to be 100% efficacious in

the 7,651 people enrolled [26]. When a case of EVD was confirmed, contacts and contacts of

contacts were randomized to either immediate vaccination or delayed vaccination (21 days later).

None of the participants that were vaccinated immediately developed EVD within 10 days,

compared with 23 contacts in the delayed vaccination clusters [26]. As a potential post-exposure

vaccine, rVSV-ZEBOV-GP has been administered twice after ZEBOV needle stick injuries, and

neither subject displayed EVD, although they developed fevers [400, 401]. While both ChAd3-

EBO-Z and rVSV-ZEBOV-GP are very promising vaccine candidates, individual-level

correlates of protection and durability in humans remain to be determined.

In parallel with vaccine research, a variety of experimental treatments and therapeutics were

prioritized by the WHO for further evaluation in September 2014, based on in vitro and animal

models of ZEBOV infection [6]. These were also considered for emergency phase I or phase

II/III trials during the ZEBOV outbreak. In addition to intensive supportive care, treatments

included: convalescent plasma [20], antisense siRNA drugs [27], monoclonal antibodies [19],

and type I interferon regimens [22]. Without an approved specific therapy, intensive supportive

care became the benchmark treatment by providing intravenous fluids, electrolyte solutions, and

57

oral rehydration to help maintain blood volume [16]. Convalescent plasma, whereby plasma

from ZEBOV survivors was transfused twice into patients during acute infection, was

administered in a non-randomized comparative study in Guinea [20]. However this treatment

required ABO-compatibility and optimal collection time from donors. It was determined after

the trial completed that the 84 patients received low levels of neutralizing anti-ZEBOV IgG

antibodies from the transfusions [402]. Moreover, convalescent plasma transfusions were not

easily amendable to sparsely equipped ETCs, and have yet to demonstrate clinical efficacy [20].

Stable Nucleic Acid Lipid Particles (SNALPS) were developed by Tekmira to deliver siRNA in

vivo, to target three ZEBOV mRNAs encoding for RdRP L, VP24 and VP35 proteins [403]. The

treatment, TKM-Ebola, could protect 100% of rhesus macaques three days post-exposure to a

lethal challenge of EBOV-Makona [21]. However, TKM-Ebola failed to demonstrate efficacy in

humans in a phase II, single-arm trial, despite infusions being well tolerated [27]. In addition,

siRNA strategies are viral strain and variant-specific, which may not be effective in future

outbreaks of EBOV [25]. As another strategy, the Public Health Agency of Canada and Mapp

Biopharmaceuticals jointly created a cocktail of three monoclonal antibodies called ZMapp,

which was administered to ZEBOV patients in a randomized, controlled phase I trial of 71

patients in Liberia, Guinea, Sierra Leone and the USA [19]. ZMapp plus standard care did not

show improved efficacy over standard care alone. The antibody cocktail has also been

administered for emergency use in seven ZEBOV patients, of which five have survived [404].

However, the slow production of antibodies from tobacco plants limits the usefulness of ZMapp

in future EBOV outbreaks. Recombinant interferon supplementation was also considered for

treatment because ZEBOV infection is associated with strong downregulation of type I

interferons α and β [405, 406]. IFN- α and IFN- β normally clear infection by activating

apoptosis in infected cells and recruiting cytotoxic cells [407]. The single-arm, historically

controlled phase II trial testing recombinant interferon β recently completed in Guinea. Nine

ZEBOV patients received daily subcutaneous injections of IFN- β-1a, and showed higher

survival (67%) when compared with 21 patients who received supportive care alone (19%) [22].

While these results are encouraging, enrollment was limited due to potential risk of interferon

treatment exacerbating EVD symptoms [22]. Taken together, these therapeutic approaches

demonstrate the logistical hurdles of amending an efficacious and well-tolerated strategy that

58

works well in small animal and NHP models of EBOV infection, with practical challenges of

implementing a clinical trial during an ongoing EBOV outbreak: patient recruitment changes

rapidly with local transmission rates, resources can be limited, electricity is often intermittent,

and limited personnel who are qualified and trained to safely administer therapies with needles in

an ETC. For all of these reasons, oral drugs that directly inhibit ZEBOV replication that are safe,

stable in warm climates, affordable, and in abundant supply, were strongly considered for

prioritization by the WHO during the ZEBOV outbreak in West Africa [6].

59

1.3.3 Repositioning Nucleoside Analogues for ZEBOV Inhibition

Nucleoside and nucleotide analogues are one of the most successful classes of antivirals for

treating HIV-1, Hepatitis B Virus (HBV), Herpes Simplex Virus (HSV), or Varicella Zoster

Virus (VZV) in patients [408-412], and are under clinical evaluation for treating HCV, CMV or

influenza virus infections [413, 414]. Pharmacological advantages include: direct inhibition of

viral polymerases that transcribe either DNA or RNA, long cellular half-lives due to sequential

phosphorylation of nucleoside prodrug to an active nucleotide triphosphate by cellular kinases,

and broad tissue distribution [415-417]. While viral and human polymerases typically have

selectivity to either Nucleotide Triphosphates (NTPs) or dNTPs, based on the ribose or

deoxyribose sugar bound to a nitrogenous base, the ability of synthetic nucleotide analogues to

disrupt nascent DNA or RNA chain synthesis is empirically determined for each viral

polymerase. Unfortunately the broad-spectrum antiviral ribavirin, active against many RNA

viruses that cause hemorrhagic fever, is a weak inhibitor of ZEBOV in vitro. Thus the ZEBOV

outbreak in West Africa accelerated the development of many nucleoside/nucleotide analogues

for the potential treatment of EVD, namely brincidofovir, favipiravir, BCX4430 and GS-5734 [6,

18, 24, 25, 418, 419].

Brincidofovir (BCV)

Brincidofovir, a broad-spectrum antiviral developed by Chimerix, is a covalent lipid conjugate of

the nucleotide cidofovir (CDV), a cytosine monophosphate analogue [25, 420]. Cidofovir is an

Acyclic Nucleotide Phosphonate (ANP), cautiously administered intravenously to treat CMV

infections of the eye in HIV-1 patients [421]. To enhance bioavailability by imitating the

digestion of monoacyl phospholipids, CDV has been conjugated to the lipid 3-hexadecyloxy-1-

propanol (HDP), making it BCV [422]. This has been found to increase the cellular uptake and

tissue distribution of orally administered BCV, with improved safety profile over CDV [423].

BCV associates with phospholipids at the cell membrane where HDP becomes cleaved, releasing

CDV into the cytosol [424]. Intracellular CDV then becomes phosphorylated twice by cell

enzymes to become CDV diphosphate (CDV-DP), the active form of the drug [425]. CDV-DP

competes with the DNA polymerase substrates of CMV and vaccinia viruses, inhibiting DNA

60

elongation by causing chain termination from its incorporation into the nascent DNA strand

[426]. BCV was originally developed to inhibit the replication of double-stranded DNA

(dsDNA) viruses, such as CMV and ADV [357]. BCV has broad antiviral activity against many

dsDNA viruses, including viruses from the families Herpesviridae, Poxviridae, Polyomaviridae,

and Adenoviridae [28]. However, long-term treatment with BCV can lead to escape mutations in

the viral DNA polymerases, selecting for BCV-resistant poxvirus or CMV [427]. In 2014, it was

discovered that BCV showed activity against ZEBOV replication in vitro, the first example of

this drug inhibiting the replication of an RNA virus. An in silico screen identifying drugs that

could be repurposed to inhibit ZEBOV replication predicted CDV may inhibit the RdRP L of

ZEBOV, suggesting a potential mechanism [428]. Yet an in vitro study on the effects of BCV

and BCV-derivatives on ZEBOV replication in cell lines only demonstrated antiviral activity of

CDV when conjugated to HDP [28]. This could suggest CDV proper has no ZEBOV antiviral

effect, or rather, that higher drug concentrations are needed in comparison with BCV. However

these two possibilities have yet to be tested, and the mechanism of inhibition of RdRP L,

potentially through RNA chain termination, has yet to be determined for either BCV or CDV.

In vivo animal studies have well documented the antiviral activity of BCV in ADV, poxvirus or

Vaccinia Virus (VACV) infections, identifying tolerated doses of BCV that protect against lethal

viral challenge and reduce disease symptoms [420, 429, 430]. However, the rapid metabolism of

BCV in non-human primates precluded the testing of BCV in gold standard models of ZEBOV

infection, complicating the design of human efficacy studies evaluating BCV for treating EVD

[25]. Nonetheless in October 2014, the FDA approved BCV for emergency use after ZEBOV

exposure. Two of three American EVD patients treated with BCV have recovered after

treatment; although their survival cannot be directly linked with BCV due to other interventions

conducted during treatment [25, 431]. Both patients were treated with a single 200 mg oral BCV

dose, and blood samples were drawn before and after 2 days of treatment [427]. Neither patient

showed genetic mutations to the ZEBOV RdRP L sequence, although the viral genome from one

patient developed a silent point mutation relative to the EBOV-Makona reference strain [427].

The third EVD patient, a man from Liberia visiting Texas and in the late stages of EVD, died

four days after initiating BCV treatment.

61

Because of advanced clinical trials testing the safety and efficacy of BCV in preventing CMV

infection [413, 432], and in vitro data of its antiviral effect against ZEBOV that has not been

made public, BCV was prioritized by the WHO in late 2014 as a compound to be evaluated in

patients with EVD [24]. The drug became fast-tracked for a single-arm, phase II ZEBOV

clinical trial that started in Liberia on January 1st, 2015 [24]. Four ZEBOV patients were

enrolled, receiving 200 mg oral BCV on day 0, followed by 100 mg oral BCV on days 3, 7, 10

and 14. Unfortunately all four patients died of illness consistent with EVD, despite no serious or

unexpected serious adverse reactions reported from BCV treatment [24]. The single-arm study

was terminated on January 31st, 2015 by the manufacturer Chimerix, before BCV efficacy could

be determined. This was due to insufficient enrollment and fewer new cases of EVD in Liberia

during the trial period [24].

Favipiravir (FPV)

Favipiravir (T-705) is another broad-spectrum antiviral, developed by Toyama Chemical Co

Ltd., to treat influenza strains that are resistant to conventional antivirals [433]. It is a purine

nucleoside analogue currently under investigation in phase III trials of uncomplicated influenza

in adults [6]. Within the cell, FPV is ribosylated and phosphorylated by cellular enzymes,

becoming the active drug favipiravir-RTP (FPV-RTP) [434]. The active drug inhibits the RdRP

of Influenza A Virus (IAV) when two molecules of FPV-RMP are incorporated consecutively

during primer extension [434]. FPV-RTP outcompetes both GTP and ATP, preventing further

primer extension and terminating RNA chain synthesis [434]. FPV has demonstrated in vitro

and in vivo activity against many RNA virus families that infect humans, including negative-

sense single-stranded RNA (-)ssRNA viruses (Orthomyxoviridae, Arenaviridae and

Bunyaviridae) and (+)ssRNA viruses (Caliciviridae, Togaviridae, Picornaviridae and

Flaviviridae) [434-440].

Two separate studies have reported activity of FPV against ZEBOV replication in vitro [23, 29].

The first study administered FPV to Vero E6 cells (African green monkey kidney epithelial) 1

hour prior to infection with ZEBOV-Mayinga, at a Multiplicity of Infection (MOI) of 0.01 [23].

Five days thereafter, infectious particles were quantified by an immunofocus assay using anti-

62

EBOV polyclonal antibodies, and cell viability measured by the MTT assay. In cell culture, FPV

was able to suppress ZEBOV infection with an IC50 of 67 μM and an IC90 of 110 μM, with no

affects on cell viability [23]. In the second report, Vero C1008 cells were infected with ZEBOV-

E718 or ZEBOV-Kikwit at a higher MOI of 0.1, and then treated immediately with FPV [29].

Infection was visualized by plaque layer formation, and FPV could inhibit ZEBOV plaque

formation with an EC50 of ~200 μM and an EC90 of ~400 μM [29], higher doses than the

previous report, which assessed FPV pretreatment and a lower ZEBOV MOI [23].

To plan for potential human studies, both reports then examined the efficacy and

pharmacokinetics FPV in small animal models of ZEBOV infection [23, 29]. This was followed

by a third study investigating the pharmokinetics of FPV in non-human primates [441]. In the

first publication, 18 IFNα/βR−/−

(A 129) knockout mice, which are immunodeficient and

susceptible to EVD, were challenged with aerosolized ZEBOV-E718, at a lethal dose equivalent

to 1 TCID50 [29]. All 6 mice given twice-daily (BID) oral FPV at 50 mg/kg starting 1 hr post-

exposure, survived for 4 weeks-post challenge, exhibiting transient weight loss but were

otherwise normal after 30 days. Conversely, the 12 mice receiving no treatment showed clinical

signs of weight loss, severe ruffling, hunched posture and blindness, and all of them died 7-8

days post-ZEBOV exposure [29]. In the second study, which used IFNα/βR−/−

(C57BL/6)

knockout mice, FPV was administered either 6 or 8 days after ZEBOV exposure [23]. Fifteen

mice were infected intranasally with 1,000 Focus-Forming Units (FFU) of ZEBOV-Mayinga,

and treated with 150 mg/kg of FPV BID, starting at 6 (N = 5) or 8 (N = 5) days post-infection

[23]. Prior to treatment, all mice lost weight rapidly, showed increasing viremia, exhibited

elevated symptoms of EVD, and a decrease in body temperature consistent with shock. In the

mice starting FPV treatment at 6 days post-infection, all 5 were able to clear infection from

blood within 4 days, and all 5 mice recovered during the 3 weeks of post-infection observation

[23]. These mice developed anti-EBOV specific antibodies, and CD8+ T-cells responding to

viral nucleoprotein, suggesting a virus-specific adaptive immune response was mounted in the

absence of type I IFN signaling [23]. In contrast, all 5 of the untreated mice died within 10 days

of ZEBOV infection [23]. Of the 5 mice starting FPV treatment 8 days-post infection, treatment

delayed death in 1/5 animals, but all of them eventually died of infection by day 15. This

63

provides evidence of a critical treatment window period where once overt symptoms and high

viremia are present, FPV may no longer be beneficial at terminal stages of EVD [23]. Lastly, a

pharmacokinetics study of daily maintenance doses of FPV (60 to 180 mg/kg BID) in uninfected

Chinese or Mauritian cynomolgus macaques, determined that a 20% dose increase would be

needed by day 7 to treat EVD, to compensate for higher drug clearance over time in larger

animals [441]. This study helped inform dosing regimens that were useful in planning EVD

clinical trials testing the efficacy of FPV.

With strong anti-ZEBOV effects shown in animal models [23, 29], acceptable safety data from

human dose-escalation trials in Japan [442], and large stocks of drug readily available, by

September 2014 FPV quickly became short-listed by the WHO for emergency use after ZEBOV

exposure. A case study of a 43-year old doctor who contracted ZEBOV in Sierra Leone, and

was treated with a combination of FPV and ZMapp in Switzerland, demonstrates the complexity

in attributing drug efficacy in non-controlled, emergency treatment settings [443]. While the

patient’s viral load decreased rapidly and the patient fully recovered, it is not clear whether

immune responses cleared infection, whether either treatment had an effect, or both [443]. To

answer such questions, international collaborations in the fall of 2014 led to the rapid planning of

the JIKI trial: a multicenter, historically controlled, single-arm phase II clinical trial in Guinea

[18]. It recruited EVD patients beginning in December 2014 and completed by April 2015.

Patients (N = 126) were offered standardized care and optional oral FPV treatment, with an

initial adult loading does of 6,000 mg FPV on the first day, and maintenance doses of 2,400 mg

FPV per day for the next 9 days [18]. Of 99 adults and adolescent analyzed, the mortality rate

was marginally reduced during the clinical trial (52.6%) compared with the historical mortality

rate at the ETC prior to the study (55%), leading to no firm conclusion of the benefits of FPV

treatment [18]. FPV was well tolerated by all patients who received it, but the study was unable

to determine the efficacy of FPV on EVD mortality or relationship with RNA viral load,

suggesting further FPV dose optimization is required [18]. Most importantly, it was observed

that baseline viremia was a strong prognostic of patient survival, regardless of treatment: the

mortality rate of those with low baseline viremia (< 107.7

genome copies/mL) was 20%,

64

compared with the 91% mortality rate of patients with high baseline viremia (> 107.7

genome

copies/mL) [18].

After the trial ended, analysis of plasma FPV concentrations from 66 of the treated patients

revealed that by day 4 of treatment, the mean plasma concentration dropped and was much lower

than anticipated by modeling: 64.4 μg/mL predicted versus 25.9 μg/mL observed [444]. Thus,

the JIKI trial did not achieve the target plasma drug exposure level defined before the trial began.

Previous pharmacokinetic research in healthy Japanese volunteers shows that single-dose FPV

has a short half-life of 2-5.5 hrs [442]. Nevertheless, a retrospective clinical trial case series

provides optimism for further FPV efficacy research in treating EVD [445]. In a Sierra Leone

hospital, 85 EVD patients were enrolled into supportive therapy (control group), while 39

patients were given oral FPV in addition to supportive therapy [445]. Relative to the control

group, patients adminstered FPV showed greater symptom improvement, reduced viral load,

longer average survival time (46.9 days vs. 28.9 days) and a higher overall survival rate (56.4%

vs. 35.3%) [445]. These are compelling reasons to design future randomized controlled trials to

test the efficacy of FPV in treating EVD, with careful optimization of drug doses to achieve

target FPV plasma levels. Currently a phase II, dose-escalation trial (FORCE) is recruiting male

Ebola survivors in Guinea, to evaluate whether high doses of FPV can reduce ZEBOV RNA

shedding in semen.

BCX4430

BCX4430 is an experimental adenosine analogue created by BioCryst Pharmaceuticals, studied

for its inhibition of RNA viruses, such as HCV, Middle East Respiratory Syndrome Coronavirus

(MERS-CoV) and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) [6]. Like

FPV, BCX4430 can broadly inhibit (-)ssRNA viruses (Orthomyxoviridae, Arenaviridae,

Paramyxoviridae and Bunyaviridae) and (+)ssRNA viruses (Filoviridae, Togaviridae,

Picornaviridae Flaviviridae and Coronaviridae) [418, 446, 447]. It is highly selective for viral

RNA polymerases, and has not been shown to incorporate into human RNA or DNA, when

human Huh-7 cells are treated with BCX4430 [418].

65

BCX4430 was originally identified from a small-molecule library of inhibitors designed to target

RNA polymerase and reverse transcriptase activity. Inside a cell it is converted to BCX4430-

triphosphate (BCX4430-TP), where after pyrophosphate cleavage, becomes incorporated into the

nascent viral RNA strand [418]. Its mechanism of action is through non-obligate RNA chain

termination, causing premature termination of viral replication or transcription. This has been

demonstrated for the HCV RNA polymerase in cell-free experiments, where BCX4430-TP

caused premature termination of RNA chain synthesis two bases after BCX3330-MP

incorporation into the growing RNA chain [418]. Within various cell lines and primary human

hepatocytes, BCX4430 becomes rapidly phosphorylated within hours [418]. In HeLa cells

transfected with an EBOV minigenome, which creates transcription and replication competent

virus-like particles (trVLPs), viral glycoprotein-GFP expressed on the cell surface was inhibited

by BCX4430 adminstered 5 hrs post-transfection, with an EC50 of ~ 12.5 μM. No adverse

effects on cell viability were detected by the lactate dehydrogenase (LDH) release assay [418].

In Hela cells infected with EBOV-Kikwit, surface expression of viral GP1,2 was completely

inhibited by BCX4430 at concentrations greater than 100 μM, with an EC50 of ~ 11.8 μM. This

was repeated in primary human monocyte-derived macrophages infected with EBOV-Kikwit and

treated with BCX4430 (EC50 of ~ 32 μM) [418]. Taken together, these in vitro findings suggest

BCX4430 inhibits ZEBOV replication; however, they only come from a single report, and have

yet to be reproduced independently.

The pharmacokinetics and efficacy of BCX4430 have been tested in both a small animal model

and non-human primate models of ZEBOV infection [418, 448]. While the prodrug has been

found to rapidly decrease in the plasma of rodents after intramuscular injection (half-life of 5

minutes), the active metabolite BCX4430-TP has an intracellular half-life of 6.2 hours in the

liver of rats [418]. In an experiment with immunocompromised C57Bl/6 mice, 30 mice were

injected daily with 0.9% saline vehicle (N = 10) or administered 150 mg/kg BCX4430 BID,

either by intramuscular injection (N = 10) or orally (N = 10), starting 4 hours prior to infection.

The mice were then infected with a mouse-adapted strain of ZEBOV-Mayinga at 1,000 PFU

[418]. All mice administered saline vehicle died within 8 days. Meanwhile, intramuscular

injections of BCX4430 provided 100% protection against lethal ZEBOV challenge, and all mice

66

lived for the 14 days of follow-up [418]. Oral administration of BCX4430 was also protective,

with 80% of the mice surviving until day 14. In a separate dose-range study in cynomolgus

macaques, 3.4 to 16 mg/kg of BCX4430 BID was injected intramuscularly into animals 48 hours

post-ZEBOV exposure [448]. Despite the 16 mg/kg dose group showing a significantly

prolonged lifespan relative to the control group (13.2 days vs 7.2 days), none of the animals

survived [448]. Thus, a second study was performed with higher doses, administered closer to

the time of infection. Rhesus macaques were injected with 16 mg/kg BID (N = 6) or 25 mg/kg

BID (N = 6) of BCX4430, or injected with saline vehicle (N = 3) after 30-60 minutes of lethal

ZEBOV challenge [448]. Following 14 days of follow-up, all animals administered 25 mg/kg

BID of BCX4430 survived, while 4/6 animals in the 16 mg/kg BID group survived, and all 3

animals in the control group died by day 9 [448]. The mean peak viral load on day 8 also

decreased in both groups of BCX4430-treated animals. Clinical trial data has yet to be reported

for BCX4430 in humans, however, a phase I study of the safety, tolerability and

pharmacokinetics of daily intramuscular injection of BCX4430 in healthy participants is ongoing

[448].

GS-5734

Recently, Gilead has reported that a novel adenosine analogue, GS-5734, inhibits ZEBOV

replication with high in-vitro efficacy [6]. GS-5734 is a monophosphoramidate prodrug with

broad-spectrum activity against (+)ssRNA viruses (Filoviridae and Coronaviridae), (-)ssRNA

viruses (Paramyxoviridae and Arenaviridae), but not against (+)ssRNA Togaviridae or

retroviruses such as HIV-1 [417, 449]. It has been shown to have low toxicity in primary human

cells and cell lines, with selectivity against viral polymerases [417].

GS-5734 was discovered from a library of nucleoside and nucleoside phosphonate analogues, in

a ZEBOV collaboration between the CDC and United States Army Medical Research Institute of

Infectious Diseases (USAMRIID) [419]. Focused screening of the ~ 1,000 compounds

identified a 2-ethylbutyl l-alaninate phosphoramidate parent drug, which through further

optimization by structure activity relationships, led to identifying an isomer that inhibited

ZEBOV in human macrophages with an EC50 = 86 nM, called GS-5734 [419]. Nucleoside

67

analogue drugs typically have slow kinetics during the first phosphorylation event, whereas GS-

5734 was designed to have a monophosphate promoiety, to bypass this rate-limiting step and

greatly enhance intracellular triphosphate concentrations of the compound [417]. GS-5734-TP

has shown to have a long half-life of 24 hrs in primary human monocyte-derived macrophages,

30-times greater when compared to its parent drug [417]. When GS-5734 was added to HeLa or

HFF-1 cells 2 hrs prior to infection with ZEBOV-Makona (5 PFU) or ZEBOV-Kikwit (0.5 PFU),

it could markedly inhibit infection measured by immune-staining 2 days later, with low EC50

values: ~0.2 μM for inhibiting ZEBOV-Makona and ~ 0.16 μM for inhibiting ZEBOV-Kikwit

[417]. In similar experiments, GS-5734 could inhibit ZEBOV-Makona infection in pretreated

primary human macrophages (EC50 = 0.086 μM) and in the hepatocyte cell line Huh-7 (EC50 =

0.07 μM) [417]. At doses greater than 0.1 μM, pretreated Huh-7 cells infected with ZEBOV-

Makona for 3 days demonstrated a GS-5734 dose-dependent reduction in viral RNA produced in

infected cells. Consistent with the mechanism of action of FPV and BCX4430, GS-5734 causes

premature chain termination during RNA synthesis of a viral RdRP, as demonstrated by its cell-

free inhibition of the RNA polymerase isolated from Respiratory Syncytial Virus (RSV) [417].

GS-5734 was also found not to inhibit human RNA Pol II or human mitochondrial RNA

polymerase, bolstering its selectivity to viral RNA polymerases [417].

In non-human primate models of ZEBOV infection, GS-5734 has exhibited unique properties

that make it a strong candidate for further development [417]. Intravenous administration of

10 mg/kg of GS-5734 to rhesus macaques lead to rapid elimination of the prodrug (half-life of

0.39 hours), where as intracellular GS-5734-TP was persistent in PBMCs (half-life of 14 hours)

[417]. Tissue distribution of the drug administered to cynomolgus macaques shows that it can

reach viral sanctuary sites (testes, epididymis, eyes, and brain) within 4 hours [417], suggesting

GS-5734 could be useful for Ebola survivors, who continue to shed virus or suffer from post-

Ebola virus syndrome [362]. Efficacy studies of GS-5734 in rhesus macaques found that daily

intramuscular injection of 3 mg/kg or 6 mg/kg of drug, starting 30-90 minutes following ZEBOV

challenge, were suboptimal in reducing systemic viremia or improving animal survival [417].

However, animals given a 10 mg/kg loading dose of GS-5734 three days-post ZEBOV exposure,

followed by daily GS-5734 injections of either 3 mg/kg (N = 6) or 10 mg/kg (N = 6), showed

68

100% survival after 28 days [417]. All animals administered saline vehicle died by day 9, and

the rhesus macaques given 10 mg/kg of GS-5734 daily showed the lowest levels of plasma viral

RNA, with no evidence of genetic variants that would indicate selection of GS-5734-resistent

virus [417].

Similar to BCV and FPV, GS-5734 is also reserved for emergency use after high-risk exposure

to ZEBOV. During the 2014-16 West Africa ZEBOV outbreak, there were two instances of the

compassionate use of GS-5734 [450, 451]. The first case was a female nurse from Scotland who

contracted ZEBOV in Sierra Leone, and was readmitted to hospital 9 months after suffering

acute meningitis and EVD relapse, with detectable virus found in her cerebrospinal fluid [450].

She was treated with corticosteroids and GS-5734 for 14 days, until her viral RNA load became

undetectable [450]. The second case was a newborn girl in Guinea, who was diagnosed with

EVD the day she was born from a ZEBOV-positive mother diagnosed with EVD [451]. The

newborn was promptly administered ZMapp, plasma from an Ebola survivor and GS-5734.

After 20 days of treatment, she showed no detectable ZEBOV RNA in her blood, and was

discharged by day 33 in good health [451]. This newborn girl is the first documented case of a

patient surviving vertical transmission of ZEBOV.

Given the propensity for GS-5734-TP to accumulate in mononuclear cells, the primary target

cells of ZEBOV infection [417], this drug may be valuable for post-exposure prophylaxis. The

large-scale manufacturing of GS-5734 permitted for phase I clinical trials evaluating the safety

and pharmacokinetics of single and multiple doses of GS-5734, administered by intravenous

infusion [419]. No serious adverse effects to GS-5734 have yet to be observed. In addition, the

distribution of the drug to viral sanctuary sites prompted interest in evaluating GS-5734 for

efficacy in treating post-Ebola syndrome, and to potentially reduce persistent viral shedding in

the male genital tract [419]. Adult male Ebola survivors are currently being enrolled in a double-

blind, randomized, placebo-controlled phase II study (PREVAIL IV), to assess whether 100 mg

of daily GS-5734 treatment can cause long-term clearance of ZEBOV and reduce viral shedding

in semen [419].

69

1.3.4 Testing Other Potent Nucleoside/Nucleotide Analogues: Zidovudine,

Lamivudine and Tenofovir

The rapid development of preclinical research and EVD clinical trials to test the efficacy of

brincidofovir, favipiravir, BCX4430 and GS-5734, demonstrate the escalated interest in

nucleoside and nucleotide analogue inhibitors of the ZEBOV RNA-dependent RNA polymerase

L. Since the West Africa ZEBOV outbreak began in 2014, several compounds approved for

other indications have shown in vitro activity against ZEBOV replication [6, 452-454]. In this

section, the tertiarty structure of RdRP L will be compared with other related viral polymerases,

allowing for comparisons to be drawn between nucleoside/nucleotide analogues and NTP

binding to RdRP L [455, 456]. This will provide the rationale for considering potent nucleoside

analogue inhibitors of HIV-1 and HBV, such as zidovudine (AZT), lamivudine (3TC) and

tenofovir (TFV), for testing of antiviral activity against ZEBOV replication. The case for

investigating various combinations of nucleoside analogues targeting the RdRP L of ZEBOV

will also be made.

Modeling ZEBOV RdRP L and nucleotide binding

Given the lack of a defined crystal structure of the EBOV polymerase, two separate modeling

studies have used the tertiary structures of related viral RNA polymerases and reverse

transcriptases [455], as well as combined homology and ab initio modeling [456], to describe

putative domains of the RdRP L of ZEBOV. The crystal structures of nucleotide binding sites

on RNA and DNA polymerases are of particular interest, as no polymerase has been shown to

have absolute template or substrate specificity [457-459]. Moreover, available structures of

polymerases, bound either to natural nucleotides or nucleotide analogues, demonstrate similar

conserved binding mechanisms [460-462].

In the first study, the ZEBOV protein L sequence together with the crystal structures of 20 RTs

from retroviruses and monomeric RdRPs from (+)ssRNA, (-)ssRNA and dsRNA viruses, led to

a model of the tertiary protein structure of RdRP L [455]. The general structure of viral RNA

polymerases and retroviral reverse transcriptases in Figure 1.12 are from [455]. The crystal

70

Fig. 1.12: General structure of polymerases from the three main groups of RNA viruses

and retroviruses. The conserved fingertips (orange), fingers (yellow), palm (green) and thumb

(red) subdomains of four prototypical viral polymerases are shown. (A) Poliovirus RNA-

dependent RNA polymerase. (B) Influenza A virus RNA-dependent RNA polymerase. (C)

Bacteriophage Φ6 RNA-dependent RNA polymerase. (D) HIV-1 RNA-dependent DNA reverse

transcriptase [455].

71

structures of 20 polymerases from four of the main viral classes share a right-hand appearance,

with three characteristic subdomains called the fingers, palm and thumb domains [463]. The

thumb domain participates in non-specific interactions with the primer strand, while the palm

domain coordinates the phosphoryl transfer reaction of an incoming ribonucleotide to the

growing RNA chain [464]. A common mechanism of nucleotidyl transfer is used by RdRP

enzymes within the palm subdomain. In this region, a conserved aspartate residue coordinates

with two divalent metal ions to catalyze the polymerization reaction of the growing RNA chain

[465, 466]. Consequently, this area of the palm subdomain is the most conserved sequence of all

viral RdRP. In many cases, a fourth fingertips domain can be present, creating a more closed

right-hand structure [455]. This fingertips domain is known to influence the interaction between

an incoming nucleotide and the template strand [467].

Specifically within the family of RdRPs, six conserved structural motifs have been identified

within the palm subdomain (labeled A-F), containing key residues for proper ribonucleotide

binding and protein conformational changes [468-470]. Key residues within the active site have

several interactions with an incoming nucleotide. Conserved aspartic acid residues within motifs

A and C of the palm subdomain, along with basic residues in motif F of the fingers subdomain,

specifically interact with the triphosphate moiety of the incoming nucleotide [468, 471]. An

aspartic residue in motif A and an asparagine in motif B of the palm subdomain also interact

with the 2’OH moiety of ribonucleotides, providing selection for ribonucleotides in the

nucleotide binding pocket of RdRPs [471, 472]. Residues in motifs A and B of the palm and

fingers subdomains of RdRPs also interact with the sugar moiety of the nucleotide, playing an

additional role in substrate discrimination [468]. Unique to RTs and DNA-dependent DNA

polymerases is a bulky side chain that creates a steric barrier preventing ribonucleotides from

entering the nucleotide binding pocket [473]. Lastly, the RNA template and primer bases

provide many interactions with the incoming nucleotide base moiety in RdRPs, strongly

influencing ribonucleiotide specificity.

Considered together, the four conserved subdomains of viral RNA and DNA polymerases, and

six highly conserved structural motifs within the palm subdomain, allowed for homology-based

construction of a general three-dimensional model for ZEBOV L (see Figure 1.13, from[455]).

72

Fig. 1.13: Predicted structure of the RdRP L of ZEBOV. (A) Alignments of other viral

polymerases suggest conserved fingers (yellow), palm (green) and thumb (red) domains are

present in polymerase L, with 90% confidence. (B) Close-up of the enzyme active site identifies

viral polymerase motifs A-E in the moded of RdRP L [455].

73

The protein L model was identified to contain the finger, palm and thumb subdomains, with

conserved structure in the palm subdomain, consistent with other viral RNA and DNA

polymerases [467, 474]. It also showed similar structural motifs with the crystal structures of

DNA and RNA polymerases bound to nucleotides or nucleotide analogues, suggesting similar

binding mechanisms may be involved [455]. These conserved regions, in particular the palm

subdomain which contains the residues for nucleotide binding, could explain why FPV and

BCX4430 exhibit broad-spectrum inhibition of many (+)ssRNA and (-)ssRNA viruses, and why

BCV was recently found to have activity against RNA viruses [455]. Thus, nucleotide analogues

designed to inhibit the active site of polymerases from different viruses, such as 3TC inhibiting

HIV-1 RT, may also inhibit ZEBOV RdRP catalytic activity [455].

In the second study, a 3-D model of the middle polymerase domain of ZEBOV protein L (AAs

648-1457) was generated by homology-mediated modelling, based on the known X-ray

structures of the viral RNA polymerases of human Enterovirus (EV), Bovine Diarrhea Virus

(BVDV) and Foot-and-Mouth Disease Virus (FMDV) [456, 475-477]. This model of the middle

domain of protein L shared significant architectural similarities with the polymerases of other

RNA viruses, namely Poliovirus, Rotavirus, HCV and Rhinovirus [456]. To determine the

putative ribonucleotide binding pocket, the crystal structures of EV and BVDV polymerases

complexed with GTP substrate were superimposed on the predicted protein L structure [456]

Complementing this approach, the solved structure of FMDV polymerase complex with the

nucleotide analogue ribavirin and template-primer RNA, also helped to identify a helical motif at

AAs 983-990 as the probable ribonucleotide binding domain: FLRQIVRR [456].

Next, with the predicted structure of this nucleotide binding domain, and conformational

information of nucleotides (ATP, CTP, GTP and UTP) and select nucleotide analogues, in silico

molecular docking studies of polymerase-ligand complexes and their associated interaction

energies could be modeled [456, 478]. The putative nucleotide binding domain appears to have

two entry sites, one on the “left” and one on the “right”. The lowest total interaction energy for

each natural nucleotide suggests that all four NTPs bind the FLRQIVRR motif from the “left”

(ATP = -8.83 kcal/mol, CTP = -11.59 kcal/mol, GTP = -9.23 kcal/mol and UTP = -8.96

kcal/mol) (Figure 1.14, from [456]). Interestingly, triphosphorylated nucleotide analogues were

74

Fig. 1.14: Model of ribonucleoside triphosphates in complex with RdRP L. Each of the four

nucleotides (green) were found to bind the “left” side of the putative nucleotide binding pocket

of ZEBOV polymerase L (yellow). (A) ATP. (B) CTP. (C) GTP. (D) UTP [456].

75

found to be either “left-handed” or “right-handed” in terms of their lowest interaction energy

with RdRP L. This separated nucleotide analogues into two distinct docking groups. For

instance, abacavir-TP (-10.42 kcal/mol), favipiravir-RTP (-11.51 kcal/mol) and BCX4430-TP (-

8.23 kcal/mol) were predicted to bind from the “left”, while tenofovir-DP (-12.51 kcal/mol) is

modeled to bind from the “right” (see Figure 1.15 for an example of nucleotide analogues

binding differently, from [456]). Moreover, these total energy interaction values of

triphosphorylated nucleotide analogues were very similar to the natural NTPs, suggesting they

could compete for substrate binding to RdRP L [456]. As an example, the model predicted FPV-

RTP would outcompete binding of CTP or UTP to the putative nucleotide binding pocket, while

BCX4430-3P would outcompete CTP binding [456]. Potential interactions between AZT, 3TC

and TFV with the nucleotide binding pocket were also described [456]. In the following three

sections, the established interactions of AZT, 3TC and TFV nucleoside/nucleotide analogues

binding and inhibiting viral RdRPs and RTs, their clinical indications for treating other viral

infections, and potential for inhibiting the RdRP L of ZEBOV, will be discussed.

Zidovudine (AZT)

In 1985, it was discovered that the thymidine analogue zidovudine, also known as

azidothymidine or retrovir, could inhibit HIV-1 replication in vitro [479]. Originally designed to

target DNA synthesis in breast cancer, AZT became the first antiviral FDA-approved for the

antiretroviral treatment of HIV-1/AIDS in 1986 [480]. AZT shows strong synergy with 3TC in

inhibiting HIV-1, and was an important component of first-line cART regimens [481, 482].

As with most other nucleoside analogues, intracellular AZT becomes phosphorylated to its active

form (AZT-TP) by host cell enzymes [483]. AZT-TP inhibits HIV-1 reverse transcriptase when

it is incorporated into the nascent DNA chain, causing early chain termination [483]. It is a weak

inhibitor of cellular DNA polymerases α and β, but can inhibit mitochondrial DNA polymerase γ

[484]. This can impair mitochondrial DNA synthesis at high AZT concentrations, causing

apoptosis of cardiac and skeletal muscle cells, which can lead to toxic mitochondrial myopathy

when used long-term in patients [485, 486]. Research into the mechanism of AZT-TP inhibition

76

Fig. 1.15: Example nucleoside/nucleotide docking to the ZEBOV RdRP L. (A) Abacavir-TP

(red) binds the FLRQIVRR motif (yellow) from the “left” side, similar to favipiravir-RTP and

BCX4430-TP. (B) Tenofovir-DP (red) binds the nucleotide binding pocket from the “right”

side [456].

77

of HIV-1 RT revealed that the enzyme undergoes isomerisation in a two-step binding reaction

with template-primer, before binding free dNTP substrate [487]. Incorporated AZT-MP into the

DNA strand inhibits the ribonuclease H (RNase H) cleavage activity of HIV-1 RT, acting as a

competitive substrate inhibitor when RT is in the presence of divalent cation activator Mn2+

, or

as an uncompetitive substrate inhibitor in the presence of Mg2+

[488]. This suggests the

conformation of RNase H is dependent on the divalent cation present, altering its mode of

hydrolysis between endonucleolytic and exonucleolytic cleavage [488].

Research into HIV-1 RT resistance mutations towards AZT treatment provides valuable insight

into the mechanisms of this drug, suggesting potential means of how AZT might inhibit the

RdRP L of ZEBOV. Mutations in the RNase H primer grip region have shown to enhance

resistance to AZT, by decreasing template switching, altering the balance between template RNA

degradation and nucleotide excision, and changing RT interactions with the template-primer

complex [489]. While other RT mutations also permit HIV-1 to overcome AZT monotherapy

and increase viral replication, substitutions at amino acids D67N, K70R, T215Y, and K219Q

come at the cost of processive DNA synthesis [490]. HIV-1 RT resistant to AZT inhibition also

show mutations that enhance the excision of AZT-MP from the end of the primer strand at the

nucleotide binding site [491]. These mutations increase AZT-MP excision because of a new

binding site that can recruit ATP, which acts as the pyrophosphate donor for AZT-MP-mediated

excision [492].

In addition to HIV-1 antiviral activity, AZT has been found to inhibit the replication of a variety

of retroviruses, DNA viruses and RNA viruses [493-496]. AZT can inhibit the replication of

HBV in HepG2 cells, a virus encoding a DNA-dependent DNA polymerase [493]. The

polymerase of HBV can transcribe from RNA as well as DNA templates, and AZT can inhibit in

vivo HBV replication in chronically infected patients [494]. Furthermore, in vitro AZT treatment

of uninfected PBMCs can prevent infection from Human T-cell Lymphotropic Virus type I

(HTLV-1) [495]. The HTLV-1 genome is ssRNA, similar to HIV-1, and encodes a reverse

transcriptase enzyme. Human Foamy Virus (HFV), another retrovirus that occasionally infects

humans, can also be efficiently inhibited with AZT treatment [496].

78

With respect to RNA viruses that cause hemorrhagic fever, AZT has also shown strong antiviral

activity [497, 498]. Hantaan Virus (HTN) is a (-)ssRNA virus that causes hemorrhagic fever

with renal syndrome (HFRS) in humans [499]. The virus is endemic to China and the Korean

Peninsula [500, 501]. An in vitro study of inhibitors of HTN found that cells treated with AZT

24 hrs post-infection, showed no detectable HTN RNA by qRT-PCR [497]. In addition, a study

of an aryl phosphate derivative of AZT, called zidampidine, showed this compound could protect

mice from lethal Lassa virus challenge [498]. Lassa virus has a bisegmented, ambisense ssRNA

genome [502]. In the zidampidine pilot study, CBA mice were injected with 250 mg/kg of

zidampidine prior (-24 hr), during (1 hr) or post-challenge (24, 48, 72 or 96 hr) of intracerbral

injections of Lassa virus (Josiah strain) [498]. The zidampidine-treated mice showed

significantly higher survival compared to mice administered vehicle [498].

From the model of nucleotide analogue docking to the putative nucleotide binding pocket of

ZEBOV RdRP L described in the previous section, it was predicted AZT-TP would have a

similar interaction energy to the viral polymerase (-11.47 kcal/mol) as natural NTPs (-8.83 to -

11.59 kcal/mol) [456]. Moreover, AZT-TP belongs to the group of nucleotide analogues that

would dock at the “right” side of the nucleotide binding motif, opposite of FPV and BCX4430

[456]. AZT-TP was also predicted to outcompete CTP binding directly, providing a potential

mechanism of this drug during in vitro ZEBOV infection [456].

Lamivudine (3TC)

Lamivudine is a nucleoside analogue of cytidine that can inhibit HIV-1 and HBV replication,

used for salvage therapy when treating HBV [503-505]. Similar to AZT-TP, the active

metabolite 3TC-TP inhibits HIV-1 reverse transcriptase when 3TC-MP becomes incorporated

into the growing DNA chain, causing early chain termination [506]. Also similar to AZT, 3TC

is a weak inhibitor of human DNA polymerases α and β [506]. 3TC is orally administered, has

high bioavailability, and may cross the blood-brain barrier [507]. 3TC is a component of cART

regimens to treat HIV-1 in patients, and is highly synergistic in inhibiting HIV-1 when combined

with AZT [408, 481].

79

In 1992, 3TC was first reported to be a potent and highly selective inhibitor of HIV-1 and HIV-2

replication in CD4+ T-cell lines and human PBMCs ex vivo [505]. It was then determined in

HeLa cells that 3TC-TP was a direct competitor of dCTP in the RNA-dependent DNA

polymerase activity of HIV-1 RT [506]. 3TC-TP can also outcompete dCTP in the presence of

human DNA polymerase γ, with 3TC-MP becoming incorporated into mitochondrial DNA

[508]. However, this enzyme has substrate exonuclease activity that can remove 3TC-MP from

the dsDNA, likely explaining the low levels of mitochondrial toxicity associated with 3TC when

compared with AZT [508].

Interestingly, HIV-1 variants that become resistant to 3TC also show reduced processivity

similar to AZT-resistant variants; however, it is at a cost to viral replication in infected primary

cells [509]. Often these mutations occur at the catalytic core of RT in a conserved YMDD motif,

where the methionine residue becomes replaced with isoleucine (M184I), and then valine

(M18V4) in subsequent HIV-1 quasispecies [509]. Under limiting dNTP concentrations, HIV-1

RT enzymes carrying either mutation produce shorter cDNA transcripts when compared with

wild type RT [509]. The decreased processivity of M184I and M184V RT mutants, slower

primer extension rate, and increased strand transfer activity can all be attributed to defective

dNTP utilization [510]. These alterations are not caused by changes in binding of the DNA

primer-RNA template or RNAse H ribonuclease activity. Thus slower cDNA synthesis, coupled

with normal dissociation rate of the primer-template, accounts for the lower processivity and

shorter HIV-1 cDNA transcripts exhibited in 3TC-resistant HIV-1 strains [510]. HIV-1 RT

enzymes carrying the M184I and M184V substitutions also demonstrate increased fidelity, and

are more accurate than wild type RT in assays testing misinsertion and mispair extension

efficiency [511]. This suggests a trade-off in 3TC-resistent RT enzymes: increased polymerase

fidelity comes at the cost of slower cDNA synthesis and reduced synthesis of full-length viral

cDNA [511]. Indeed, HIV-1 carrying the RT M184V mutation, which produces more accurately

transcribed viral cDNA, is less capable of compensatory mutagenesis that allows reversion back

to wild type replication kinetics, impairing viral fitness [512]. The effects of substitutions at

Met184 have been further clarified with crystal structures of 3TC-TP bound to M184I mutant

HIV-1 RT, and compared with 3TC-TP bound to wild type HIV-1 RT [513]. As a beta-branched

80

amino acid, the isoleucine mutation creates steric hindrance at position 184, allowing entry of

dNTPs to the nucleotide binding pocket but not 3TC-TP [513]. The valine mutation at the same

position likely has the same effect, as it is also a beta-branched amino acid. Moreover, the

crystal structure of the M184I mutant HIV-1 RT revealed repositioning of the template-primer,

suggesting how the 3TC-resistence mutation could be interfering with optimal enzyme

polymerase activity [513].

3TC has also shown to effectively inhibit the polymerase activity of the dsDNA virus HBV and

retrovirus HTLV-1, similar to AZT [514, 515]. In experiments with duck HBV, 3TC-TP was

shown to cause DNA chain termination as a competitive inhibitor of the viral DNA polymerase,

with respect to dCTP [514]. However, development of HBV resistance mutations such as

M552V, M552I or L528M, precludes 3TC as a first-line therapy for HBV infection in patients

[516]. It appears that mutations to HBV DNA polymerase do not confer 3TC resistance through

removal of 3TC-MP by an exonuclease mechanism [517]. These mutations likely cause steric

hindrance with their side-chains, preventing 3TC-TP access to the nucleotide binding pocket,

similar to HIV-1 RT resistance to 3TC [516]. Homology modeling of the catalytic core of HBV

polymerase revealed that the mutation at Met522 was most likely to directly contribute to this

steric hindrance, where as mutation at Leu528 induced broader rearrangement of nucleotide

pocket residues [516]. 3TC treatment has also been administered to reduce HTLV-1 infection

and lessen the associated myelopathy exhibited by HTLV-1 infected patients [515]. For

instance, in a pilot study of 5 HTLV-1 patients administered 3TC, a 10-fold reduction in viral

DNA was observed [515].

The potential use of 3TC to inhibit ZEBOV replication or treat EVD patients in an emergency

context remains controversial. During the 2014-16 West Africa ZEBOV outbreak, there were

anecdotal reports of 3TC being used to treat patients with EVD in Liberia. For instance Dr.

Logan, the Chief Health Officer of Bomi County in Liberia, administered AZT or 3TC to 15

ZEBOV patients in his care, of which 13 survived [518]. However, these results have not been

published as peer-reviewed clinical case studies. For a potential mechanism of action for 3TC,

3TC-TP is predicted to interact with the putative nucleotide binding site of ZEBOV RdRP L,

comparable to FPV-RTP, BCX4430-TP and AZT-TP [456]. Modeling of 3TC-TP docking

81

suggests it would bind this region from the “left” side, resembling FPV-RTP and BCX4430-TP

binding [456]. The interaction energy of 3TC-TP with the polymerase was comparatively higher

(-7.26 kcal/mol) than other nucleotide analogues modeled in silico, suggesting weaker

competition with natural NTP binding would be predicted [456]. Indeed, the effects of 3TC in a

preliminary cell-based assay did not show activity in reducing ZEBOV-Kikwit infection in Vero

E6 cells, Hep G2 cells, or in human monocyte-derived macrophages [519]. However, these

experiments tested 3TC treatment 1 hr prior to infection, which may not be enough time for

cellular enzymes to convert the prodrug to active 3TC-TP. It has been reported in human

subjects that at 3TC oral doses of 150 or 300 mg, peak intracellular concentration of 3TC-TP in

PBMCs occured at 2.95 and 4.10 hours, respectively [415]. Moreover the in vitro ZEBOV study

of 3TC did not include a nucleoside analogue prodrug as a +ve control for ZEBOV inhibition,

but rather the drug toremifene (TOR), which destabilizes the ZEBOV GP1,2 trimer and triggers

early release of GP2, [519, 520]. Already in its active form, TOR would be expected to show

antiviral activity 1 hr prior to ZEBOV infection [519]. Accordingly, these disparate findings that

predict 3TC interacting with the ZEBOV nucleotide binding pocket [456], weak in vitro

inhibition of ZEBOV replication [519], and use to treat some EVD patients in Liberia [518],

warrant further in vitro testing of 3TC during ZEBOV replication, to clarify potential antiviral

effects of this drug.

Tenofovir (TFV)

Tenofovir disoproxil fumarate (TFV-DF) is an adenosine monophosptate nucleotide analogue,

belonging to the same class of phosphonates as BCV and CDV [521]. While TFV cannot cure

HIV-1 or HBV infection, it is used in combination with other drugs to prevent or manage HIV-1,

and ameliorate chronic HBV symptoms [522, 523]. After oral administration, TFV disoproxil

fumarate becomes absorbed in the gut and cleaved, releasing TFV into the bloodstream [521].

The phosphonate moiety of TFV allows it to be rapidly mono- and diphosphorylated to the active

drug TFV-DP in two steps, rather than three. TFV-DP inhibits HIV-1 RT activity through

premature cDNA chain termination, and shows limited inhibition of human DNA polymerase α,

β, γ [521, 524]. While viral RNA and DNA polymerases discriminate between NTPs and dNTPs

82

during RNA or DNA chain elongation, this does not occur with TFV-DP because of its absence

of a ribose or deoxyribose moiety in its bioactive structure [456].

In 1998, it was discovered that TFV selectively inhibits HIV-1 reverse transcriptase [521]. TFV

alone, or in combination with rilpivirine (RPV) and emtricitabine (FTV), can form dead-end

complexes with the RT polymerase of HIV-1, producing chain-terminated DNA primer/template

[525]. This effect was synergistically enhanced by the presence of the other two NRTI drugs,

leading to more stable chain termination and inhibition of HIV-1 replication [525]. TDF

treatment in HIV-1 patients has been successful when they harbor HIV-1 quasispecies with

substitution mutations that provide resistance to multiple NRTIs, described as the Q151M

complex [526]. This multidrug-resistant HIV-1 genome carries five characterisitic mutations in

the RT gene: A62V, V75I, F77L, F116Y and Q151M [526]. However, it has recently been

found in an HIV-1 clinical isolate that an additional RT mutation, K70Q, can add a high level of

HIV-1 resistance to TFV treatment, and only in viruses that already have the Q151M complex

background [526]. The additional K70Q mutation does not confer increased excision of TFV

from the cDNA chain, but changes hydrogen bonding patterns in the polymerase [526]. This

selectively reduces the affinity of TFV-DP to the nucleotide binding pocket of HIV-1 RT,

reducing TFV incorporation into the newly synthesized cDNA chain [526]. Another important

HIV-1 RT resistance mutation to TFV is K65R, however there is a low prevalence of sexual

transmission of various HIV-1 subtypes carrying this substitution mutation [527]. This finding

has important implications for the inclusion of TDF in PrEP regimens, as TDF reduces the

transmission of HIV-1 to uninfected people [527].

The interactions of TFV with the RT of HBV have also been extensively studied. In an open-

label study of twenty HIV-1 and HBV coinfected patients, a significant decrease in HBV viral

load was measured after 52 weeks of daily TFV treatment, as part of their cART [522]. This was

true even for 11 coinfected patients with HBV carrying the 3TC resistance sequence YMDD or

YIDD [522]. Unique to HBV DNA polymerase, its own protein sequence can initiate viral

cDNA synthesis as a protein primer [528]. It has been shown that TFV-DP strongly inhibits the

second stage of protein priming, becoming incorporated into the viral DNA primer instead of

dAMP [528]. From 2008 until 2014, no TFV drug resistance mutations have been detected in

83

clinical samples of HBV from patients treated with TFV-DF monotherapy [529]. To explain this

phenomenon, in silico modeling of the HBV polymerase suggests TFV has significantly strong

binding affinity to the nucleotide pocket of this enzyme [529]. In the HBV genome, the

polymerase gene overlaps another gene coding for surface protein. This creates an additional

genetic constraint that limits the mutational freedom that would allow for TFV drug resistance to

develop during HBV infection [529].

Little preclinical research has evaluated the potential antiviral activity of TFV or TFV-DF

against RNA viruses, including ZEBOV. Of the 20 nucleotide analogues modeled to dock to the

putative nucleotide binding domain of ZEBOV RdRP L, TFV-DP had the lowest total interaction

energy (-12.51 kcal/mol) [456]. TFV-DP was the best nucleotide/nucleoside candidate

predicted to outcompete natural NTPs binding the FLRQIVRR motif [456]. Similar to AZT-TP,

TFV-DP was modeled to bind this motif from the “right” side [456]. Given the absence of a

ribose moiety in the structure of TFV, whether TFV is capable of inhibiting the RdRP L of

ZEBOV or ZEBOV replication in living cells, deserves further attention [456].

Combination regimens to inhibit ZEBOV

When infected healthcare workers were airlifted from sites of ZEBOV exposure in West Africa

to be treated in Western hospitals, these patients had a collectively low mortality rate [25]. This

lower rate might be attributed to the multiple therapeutics administered to these patients, in

addition to intensive supportive care [431, 443, 451]. Thus, research into therapy combinations

that inhibit ZEBOV replication, such as ZMapp or TKM-Ebola formulations, are a high priority

in developing an effective ZEBOV treatment [21, 30]. In vitro and small animal model research

into combinations of IFNs with ZMapp, which may synergistically inhibit ZEBOV replication,

are underway [6]. Furthermore the JIKI trial authors surmised that in addition to optimization of

the FPV dosing regimen, the possibility of treatment combinations with FPV should also be

evaluated in future EVD clinical trials [6, 18]. This led to oral FPV and ZMapp combinations

being administered in the randomized, controlled trial PREVAIL II ,which had sites in Libera,

Guinea, Sierra Leone and the United States [19]. In addition, low doses of the nucleoside

analogue ribavirin have shown to potentiate the activity of FPV in guinea pigs challenged with

84

Junin virus, the (-)ssRNA arenavirus responsible for Argentine hemorrhagic fever [435]. FPV

and ribavirin synergistically inhibit a variety of other (-)ssRNA viruses that cause hemorrhagic

fevers: Pichinde virus in hamsters [435], Lassa virus in cell culture and in lethal mouse model of

infection [530], and mice infected with Rift Valley Fever Virus (RVFV) [436]. Hence, drug

combinations that improve the efficacy of FPV are of great interest in ZEBOV preclinical and

clinical research.

The molecular docking model of the ZEBOV nucleotide binding pocket provides additional

rationale for considering two-drug combinations for inhibition of ZEBOV replication in vitro

[456]. When nucleoside analogues preferentially dock on the “left” side of the nucleotide

binding motif (FPV-RTP, BCX4430-TP and 3TC-TP), it leaves the “right” side open for binding

of ATP, CTP, GTP, and UTP can still bind from the “left” side (see Figure 1.16, from [456]).

Likewise, nucleotide analogues predicted to bind the “right” side of the nucleotide binding motif

(AZT-TP and TFV-DP), leave entry on the “left” side for ATP, CTP, GTP and UTP binding

[456]. Thus, optimal inhibition of the nucleotide binding pocket predicts a combination of two

drugs that flank both sides of the motif, where one preferentially occupies the “left” and the other

inhibitor occupies the “right” side, to sufficiently outcompete incoming NTPs [456].

Recalculating the interaction energy in the scenario of two different nucleotide analogues

binding the enzyme, such as FPV-RTP + TFV-DP, considerably lowers their total interaction

energy [456]. It becomes -12.28 kcal/mol for FPV-RTP and -10.80 kcal/mol for TFV-DP, much

lower than the values for ATP (-4.84 kcal/mol), CTP (-6.83kcal/mol), GTP (-7.55 kcal/mol) or

UTP (-8.11 kacal/mol) [456]. A reduction of 1 kcal/mol, indicates a 10-fold higher affinity for

binding, in a simple E + S⇔ES model [456]. Thus, the interaction energies of natural NTPs for

RdRP L are significantly surpassed by specific combinations of two nucleotide analogues, which

specifically occupy both flanking sites of the nucleotide binding pocket [456]. Administration of

FPV + TFV for example, may inhibit ZEBOV polymerase activity and subsequent transcription

and replication of the viral (-)ssRNA genome. This strategy has two additional benefits over

monotherapy: 1) It could impede the rate of selection for drug resistant mutations against both

drugs, and 2) Inhibiting viral replication in vivo during the window period of high viremia days

85

Fig. 1.16: Nucleotide analogues binding ZEBOV RdRP L in the presence of NTPs. (A)

Abacavir-TP (red) docks to the nucleotide binding motif from the “left”, allowing most NTPs

(green) access to the “right” side. (B) The opposite trend is found for tenofovir-DP (red), where

it docks from the “right”, permitting NTPs to bind at the “left” [456].

86

after infection [18, 23], could provide the immune system enough relief to mount an effective

adaptive response to clears ZEBOV infection [456].

Treating retrovirus or RNA virus infections with specific combinations of nucleoside analogues

was a paradigm shift in 1996, ushering in the era of combination antiretroviral therapy for

treating HIV-1/AIDS [531]. Drug synergy can help lower plasma viremia by inhibiting multiple

viral targets, reduce drug toxicity by requiring lower drug doses, and provide a higher barrier to

selecting resistance mutations that occur with monotherapy, as previously described for HIV-1,

HBV and HCV [178, 481, 532, 533]. While routinely used to treat HIV-1, HBV or co-infection

of both viruses [410], cART has not yet been explored in preclinical ZEBOV research. This is

partly due to constraints caused by limited access to BSL4 facilities for in vitro or animal testing.

In 2014, a sophisticated new trVLP model of the ZEBOV replication lifecycle was developed,

allowing for rapid testing of therapeutics targeting any stage of the virus lifecycle in a BSL2

laboratory [534, 535]. This permitted efficient testing of drug combinations that was not

previously feasible. Furthermore, the potential mechanism of action of AZT, 3TC or TFV

during ZEBOV replication remained untested in living cells. Nucleoside analogues can disrupt

viral replication by interfering with polymerase NTP binding [506], induce RNA chain

termination [417, 426], or increase lethal mutagenesis [536], among other mechanisms [488].

Additionally, it has not been tested whether 3TC, AZT or TFV potentially synergize with leading

ZEBOV drugs candidates such as FPV, fast-tracked by the WHO for phase II/III EVD clinical

trials.

87

1.4 Statement of the Problem, Rationale, Hypotheses and

Objectives of this Work

1.4.1 Statement of the Problem

Discovering host non-receptor tyrosine kinases that facilitate HIV-1 infection, yet lessen

replication when inhibited with small-molecule inhibitors, is an underexplored area of research

that could improve conventional cART. Moreover, with no approved treatments for EVD,

repositioning drugs that directly inhibit ZEBOV replication, such as nucleoside analogues that

inhibit DNA or RNA synthesis of other viruses, may inhibit ZEBOV replication in vitro.

1.4.2 Rationale

Both c-SRC and PTK2B have been implicated during HIV-1 infection from various means of

investigation. Shortly after TCR-CD3 activation of a CD4+ T-cell, or G protein-coupled receptor

stimulation, both c-SRC and PTK2B interact and become activated [197, 293, 340]. In addition,

c-SRC and PTK2B also become activated after treating immature DCs with HIV-1 gp120 [323],

or infection of Jurkat T-cells with intact virus [214]. HIV-1 also rearranges the cytoskeleton

upon fusion and entry, and PTK2B has defined roles in T–cell cytoskeletal rearrangements at

focal adhesions [340]. Moreover JCaM1.6 T-cells, which lack functional LCK and FYN

expression but expresses c-SRC, are more infectable with HIV-1 [209]. Inhibition of the SFKs

in CD4+

T-cells with PP2 inhibited viral p24 production after 6 days [264], while shRNA

knockdown of PTK2B in T-cell lines reduced HIV-1 infection after 6 days [345]. HIV-1 Nef

binds and activates c-SRC kinase activity [320], and may be the cause of Nef-induced AIDS

phenotype in transgenic mice [164, 168]. In addition, proteins downstream of c-SRC signaling

(SAMHD1 in the cytosol, Sam68 in the nucleus) already have defined roles during HIV-1

infection [317, 537]. Furthermore, c-SRC does not contribute to Gag-mediated viral assembly

during budding [227], suggesting the effects of c-SRC signalling may occur at earlier stages of

the viral lifecycle. My thesis will attempt to answer the following questions: Are c-SRC and

PTK2B natural restriction factors of HIV-1 during early T-cell infection, or do they facilitate

88

early HIV-1 infection in T-cell lines and primary CD4+ T-lymphocytes? At which stages of the

viral replication cycle do c-SRC and PTK2B have an effect? And if one or both facilitate

infection, can FDA-approved small-molecule kinase inhibitors that target either of these NRTKs,

be used to inhibit HIV-1 infection in primary CD4+ T-cells?

My thesis will also make use of a new trVLP model of ZEBOV replication, permitting rapid

assessment of new therapies and combinations of therapies that can be tested in a BSL2 lab

environment [534, 535]. The nucleoside analogues BCV, FPV, BCX4430 and GS5734

demonstrate broad-spectrum inhibition of other RNA viruses, DNA viruses and retroviruses [6,

357, 417, 455], becoming fast-tracked by the WHO for additional ZEBOV preclinical research

[28, 418, 441], emergency phase II/III efficacy trials in West Africa [18, 24], and ZEBOV post-

exposure treatment [427, 443, 450]. Other nucleoside analogues AZT, 3TC and TFV, which

show potent inhibition of HIV-1, HBV and other (-)ssRNA viruses that cause hemorrhagic fever

[479, 493, 497, 505, 514, 521], have been predicted to bind the ZEBOV nucleotide binding motif

of RdRP L, with similar or greater affinity than natural NTPs or other nucleotide analogues

modeled [456]. Moreover, combinations of nucleotide analogues that preferentially occupy the

“left” side of the nucleotide binding pocket (FPV-RTP, BCX4430-TP and 3TC-TP) are predicted

to synergize with drugs that enter the “right” side of the binding pocket (AZT-TP and TFV-DP)

[456]. AZT, 3TC and TFV show limited inhibition of human DNA polymerases, and broad

biodistribution to immune cells and tissues where ZEBOV replication persists during acute and

chronic infection [416, 484, 508, 538]. Furthermore, anecdotal reports of patients treated for

EVD with 3TC and AZT in Liberia warrant further testing of these drugs in cell culture infection

of ZEBOV [518]. Combination regimens with FPV, the most promising drug candidate in

ZEBOV clinical trials to date [18, 19], and other experimental therapies such as IFN treatment,

should also be compared to one another to prioritize which combinations are tested against fully

infectious ZEBOV in BSL4.

1.4.3 Hypotheses

1) Inhibiting or reducing host non-receptor tyrosine kinases c-SRC or PTK2B will restrict early

HIV-1 infection in T-cell lines or primary human CD4+ T-cells.

89

2) Nucleoside/nucleotide analogue inhibitors AZT, 3TC and TFV, which demonstrate potent

antiviral activity against other RNA viruses and retroviruses, will inhibit an in vitro model of

ZEBOV replication alone or in combination with other therapies.

1.4.4 Objectives of this Work

1) Assess whether c-SRC and PTK2B facilitate HIV-1 infection in T-cell lines, by inhibiting or

reducing their expression with ADV vectors expressing dominant-negative kinase mutants, tool-

drug inhibitors or siRNA knockdown.

2) Determine whether c-SRC and PTK2B have similar roles in primary CD4+ T-cells collected

from healthy human donors, and investigate whether FDA-approved SFK inhibitors, or SFK

inhibitors in advanced clinical trials, can inhibit HIV-1 replication ex vivo.

3) Evaluate whether antivirals with established safety profiles and efficacy in inhibiting other

RNA viruses or retroviruses, can inhibit ZEBOV replication alone or in combination, in an in

vitro trVLP model of infection or with fully-infectious ZEBOV.

1.4.5 Organization of the Thesis

The strong evidence of the SFK roles during the HIV-1 replication cycle, coupled with new

kinase inhibitors with enhanced target specificity being evaluated in clinical trials outside of

cancer research, motivated the investigations contained in this thesis. Moreover drug

repurposing and combination therapy with nucleoside analogues, successful at treating HIV-1 or

HBV infection, have not been adequately investigated in ZEBOV replication in vitro.

In chapter 2, “c-SRC and PTK2B Protein Tyrosine Kinases Play Protective Roles in Early HIV-1

Infection of CD4+ T-Cell Lines,” kinase inhibitors, protein overexpression ADV vectors and

siRNA knockdown are used to determine the roles of c-SRC and PTK2B during in vitro HIV-1

infection in BSL3 containment. These T-cell line experiments laid the groundwork towards a

more comprehensive study of both kinases during HIV-1 replication in primary human CD4+

T-

cells in chapter 3; “c-SRC Protein Tyrosine Kinase Regulates Early HIV-1 Infection Post-Entry.”

90

This chapter investigates four c-SRC inhibitors (FDA-approved or under clinical investigation)

for their ability to inhibit HIV-1 infection ex vivo, using X4 and R5 lab-adapted strains and

clinical isolates of HIV-1. It was also determined at which early stage during the viral lifecycle

that is most dependent on c-SRC or PTK2B kinase expression.

In chapter 4, “A Rapid Screening Assay Identifies Monotherapy with Interferon-ß and

Combination Therapies with Nucleoside Analogues as Effective Inhibitors of Ebola Virus,”

nucleoside/nucleotide analogue inhibitors were assessed in a new model system of ZEBOV

replication in vitro. Timely during the 2014-16 West African ZEBOV outbreak, nucleoside

analogues and therapeutics fast-tracked for EVD clinical trials were compared or combined in

vitro, to assess whether they inhibit ZEBOV transcription and replication competent virus-like

particles, or fully-infectious virus tested under BSL4 conditions at the National Microbiology

Laboratory in Winnipeg.

Lastly in chapter 5, “Key Findings, Future Perspectives, Conclusions and Broader Significance,”

limitations of the present work are discussed, as well as how the findings of this thesis relate to

preclinical HIV-1 and ZEBOV drug discovery. This chapter also suggests future studies that

could investigate promising therapeutics evaluated in this thesis, to potentially inhibit viral

replication in animal models of HIV-1 or ZEBOV infection.

91

Chapter 2: c-SRC and PTK2B Protein Tyrosine Kinases

Play Protective Roles in Early HIV-1 Infection of CD4+ T-

Cell Lines.

Role of author McCarthy S.D.: Performed the experiments for Figure 2.1B, and all the

experiments for Figures 2.2-2.7. I analyzed all the data, wrote the manuscript draft, and

performed experiments to address reviewer’s comments.

A version of this chapter was published in the Journal of Acquired Immune Deficiency

Syndromes:

McCarthy, S.D., Jung, D., Sakac, D., and Branch, D.R. 2014. c-SRC and Pyk2 protein tyrosine

kinases play protective roles in early HIV-1 infection of CD4+ T-cell lines. JAIDS, 66(2): 118-

26.

92

2.1 Abstract

Background: During early HIV-1 infection of CD4+ T-lymphocytes, many host protein tyrosine

kinases (PTK) become activated within minutes, including phosphoprotein pp60c-SRC

(c-SRC)

and the focal adhesion kinase family member, protein tyrosine kinase 2 beta (PTK2B, Pyk2).

Whether their activation facilitates or impedes infection remains to be determined.

Methods: c-SRC kinase inhibitors (SU6656, PP1 and PP2), adenovectors (wild type (WT) and

dominant-negative (DN) c-SRC) or siRNA (targeting c-SRC or PTK2B) were used to inhibit,

compete with or knockdown c-SRC in Jurkat C, Jurkat E6-1, Hut 78 or Kit225 T-cell lines.

Cells were then infected with HIV-1 luciferase reporter virus expressing VSV-G or HXB2(X4)

envelope and luciferase activity was measured after 2 days. Reverse transcriptase activity and

viral cDNA were measured 1 hr post-infection, while integrated virus was measured 12 hr post-

infection.

Results: Pre-treating Jurkat T-cells with SU6656 led to increased VSV-G luciferase activity. In

the adenovector experiments, T-cells overexpressing DN c-SRC, but not WT c-SRC, showed

increased luciferase activity following VSV-G infection. siRNA knockdown of c-SRC or

PTK2B, followed by HXB2 infection in Jurkat T-cells, lead to increased reverse transcriptase

activity, viral cDNA, integrated virus, as well as increased luciferase activity.

Conclusions: PTK2B is known to interact with c-SRC. Thus PTK2B activation, which

coincides with increased c-SRC activity during HIV-1 infection, could be responsible for c-SRC

activation. Reduced c-SRC activation increases HIV-1 reverse transcription, integration and/or

transcription, suggesting the high c-SRC activity seen early in HIV-1 infection may be a cellular

response to slow or prevent early infection in CD4+ T-cells.

93

2.2 Introduction

Chronic immune activation and T-cell dysregulation persists during asymptomatic HIV-1

infection, and yet we still do not have a clear understanding of the early stages of these events

[83]. From the initial binding of viral glycoprotein 120 (gp120) to T lymphocyte receptor CD4

and chemokine coreceptors CXCR4 or CCR5, signaling cascades are induced that promote viral

infection [147]. It is known that tyrosine kinases are major regulators of HIV-1 infection

because the pan-tyrosine kinase inhibitor, genistein, inhibits HIV-1 infection in macrophages

[151]. Moreover, HIV-1-infected patients show defective early protein tyrosine phosphorylation

in peripheral blood mononuclear cells [149] and in CD4+ T-cells specifically [148]. Indeed,

much research has been done on the proximal signaling cascades induced upon viral binding and

fusion, which interfere with normal T-cell activation and cortical actin rearrangement [539].

However, a functional role for many kinases hijacked immediately following viral entry remains

to be discovered.

Non-receptor tyrosine kinases play critical roles throughout the HIV-1 lifecycle in T-cells.

Changes in tyrosine phosphorylation signaling are necessary for viral entry [209], actin

remodeling [135, 539], viral RNA reverse transcription [135], translocation of the viral pre-

integration complex (PIC) to the nucleus, viral integration [155], viral DNA transcription and

viral egress [152]. In particular, HIV-1 infected T-cells show striking changes in the activity of

the SRC-family of tyrosine kinases [214]. They become activated within minutes of HIV-1

infection; however their roles at this early time point are only partially understood. There are

eight SRC family members, four of which are expressed in T-cells: LCK, c-SRC, FYN and c-

YES. LCK has been the most studied of these four in terms of early HIV-1 infection [148, 209,

217], as it is a proximal signaling molecule directly associated with CD4 and is critical for T-cell

activation and growth [213]. Increased LCK activity was found to reduce viral replication in

various T-cell lines [209]. In addition, increased FYN activity correlates with greater

recruitment of tyrosine-phosphorylated APOBEC3G into HIV-1 particles [238], corroborating

our previous observation of higher FYN activity in patients with asymptomatic HIV-1 infection

94

[149]. Clearly the SRC family of protein kinases is implicated in HIV-1 infection, but the

functional role of activated c-SRC during early HIV-1 entry remains unknown.

Previously we have shown that negligible c-SRC protein exists in resting peripheral blood T

lymphocytes, but that it is produced within 12 hours of T-cell stimulation [293]. c-SRC

phosphorylates more than 64 known cellular substrates, activating signaling cascades involved in

cell migration, cytoskeletal rearrangement, cell proliferation and cell survival [294]. The

enzyme often associates with cell membranes, in particular at focal adhesions of the plasma

membrane and the perinuclear golgi region [189]. When HIV-1 initially attaches to the host

membrane, viral gp120 is able to bind the G protein-coupled receptor CXCR4 or CCR5,

inducing the autophosphorylation of protein tyrosine kinase 2 beta (PTK2B, Pyk2) within the

cell [147]. PTK2B plays an important role at focal adhesions at the cell periphery and in

cytoskeleton remodeling. Activated PTK2B can then recruit the binding of the c-SRC SH2

domain, which allows c-SRC to be autophosphorylated at Tyr419, activating the enzyme [197,

322]. Both of these kinases become phosphorylated within minutes of HIV-1 infection. Recent

work has shown that PTK2B shRNA knockdown in T CEM cells reduced viral p24 production

within six days of HIV-1NDK infection [345]. Moreover Gilbert et al. showed that inhibiting c-

SRC with the kinase inhibitor PP2 caused a dramatic decrease in p24 production after six days of

infection [264]. However, the roles of PTK2B and c-SRC in the first few hours of early HIV-1

infection, when these enzymes are first activated, are not well defined.

We hypothesize that c-SRC and PTK2B non-receptor tyrosine kinases play an important role in

facilitating early HIV-1 infection of CD4+ T-cells. By using c-SRC drug inhibitors, a dominant

negative c-SRC construct and siRNA knockdown, we were surprised to find that reducing c-SRC

protein levels or its activity caused an increase in viral infection in three laboratory T-cell lines.

This research is significant because small molecular kinase inhibitors targeting c-SRC may not

be efficacious in reducing initial HIV-1 infection, but may be valuable in studying the formation

and/or stability of the HIV-1 reverse transcriptase complex and pre-integration complex.

95

2.3 Materials and Methods

Cells and Cell Culture Conditions: In these in vitro experiments, we used the human T-cell

leukemia cell lines Jurkat C (from Jurkat-FHCRC, a gift from Dr. G. B. Mills, MD Anderson

Cancer Center, Houston, TX, USA), Jurkat E6-1 and a cutaneous T lymphocyte cell line Hut 78

from the American Type Culture Collection (ATCC, Rockville, USA). We also used a chronic

lymphocytic leukemia T-cell line, Kit 225, also a gift from Dr. G. B. Mills. These T-cell lines

were grown in complete medium consisting of RPMI-1640 with L-glutamine and NaHCO3

(SIGMA, St. Louis, USA), supplemented with 10% (vol/vol) heat-inactivated Fetal Bovine

Serum (FBS; Wisent, Saint-Jean-Baptiste, Canada), 100 units/mL penicillin (Gibco, Burlington,

Canada), 100 μg/mL streptomycin (Gibco), 10 μg/mL gentamycin (Gibco), and 10 units/mL

human recombinant Il-2 (Kit 225 cells only; SIGMA). Human embryonic kidney cells (HEK

293T, ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) with the

same proportion of FBS and antibiotics as above. MT-4 cells from the NIH AIDS Research and

Reference Reagent Program (Germantown, USA) were used to determine viral multiplicity of

infection (MOI). All cells were incubated at 37°C in a 5% CO2 atmosphere.

Generation of infectious HIV-1, VSV-G pseudoenveloped and HXB2 enveloped virus: In

brief, X4 HIV-1IIIB from the NIH AIDS Research and Reference Reagent Program (Rockville)

was grown in Jurkat C cells (1 x 106

cells/mL) in a biosafety level 3 (BSL3) lab [540]. The cell

supernatant was collected, viral stocks were titrated and assessed by p24 Antigen ELISA

(ZeptoMetrix, Buffalo, USA), and aliquots were frozen at -80°C. MOI was determined using

MT-4 cells [540].

We produced recombinant, replication deficient VSV-G pseudoenveloped or HXB2 enveloped

(X4) HIV-1 containing a luciferase gene, as described previously [541]. Briefly, 2.5 x 106 HEK

293T cells were plated with 10 mL of DMEM 24 hours prior to transfection. We co-transfected

15 μg of HIV-1 NL4-3luc (luciferase gene inserted into viral nef gene and env deficient; a kind

gift from Dr. Veneet KewalRamani, New York Medical University, NY) with 10 μg of VSV-G

env plasmid (amphotropic envelope of the vesicular stomatitis virus; a generous gift from Dr. M.

Tremblay, Quebec City, PQ) into each 10 cm plate of HEK 293T cells with the CalPhosTM

96

Mammalian Transfection Kit (Clontech, Mountain View, USA), following the manufacturer’s

instructions. The same was performed for the HXB2 plasmid (HIV-1 NL4-3luc lacking nef and

containing HXB2 (X4) env; another generous gift from Dr. M. Tremblay) at 15 μg per plate.

DMEM media was replaced after 25 hours and viral supernatant was collected on days 2 and 3.

The supernatant was syringed through 0.45 μm pore filters (Millipore, Billerica, USA) , then

concentrated by underlaying with a 20% sucrose gradient and centrifuging at 16,000 x g for 90

minutes at 4 °C. Virus pellet were resuspended in TNE buffer (20 mM Tris-HCl, pH 7.5, 1 mM

EDTA and 100 mM NaCl) and stored in -80°C aliquots. p24 levels were measured by ELISA.

All viruses underwent one freeze-thaw cycle prior to infection studies.

c-SRC Drug Inhibition: Prior to infection, 2 x 106 T-cells were inhibited with 5-20 μM of

SU6656 (In SolutionTM

), 10 μM PP1 (Analogue) or 10 μM PP2 (Calbiochem, La Jolla, USA) for

1 hr, washed with Dulbecco’s Phosphate Buffered Saline (PBS) lacking calcium chloride and

magnesium chloride (SIGMA), resuspended in RPMI media, and then counted again. The

solvent Dimethyl Sulfoxide (DMSO, Bioshop, Burlington, Canada) was used as a -ve control for

SU6656.

Kinase Assay: Jurkat C cells treated for 1 hr with SU6656 were lysed with

radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM HEPES, pH 7.3, 1% Nonidet P-40,

0.1% SDS, 0.1% sodium deoxycholate, 150 mM NaCl, 1 mM Na3VO4, 50 mM ZnCl2, 2 mM

EDTA and 2 mM PMSF), and immunoprecipitated with anti-c-SRC clone 327 (Oncogene,

Cambridge, USA). Lysates and beads were washed then re-suspended in kinase buffer,

including 5 μCi γ32

P-ATP (MP Biomedicals, Santa Ana, USA) and heat inactivated enolase (MP

Biomedicals) as substrate. Protein samples were boiled for 10 minutes and then separated on a

12% reducing SDS-PAGE gel and transferred to an Immobilon-P membrane (Millipore).

Enolase phosphorylation by c-SRC was determined by autoradiography. In vitro kinase activity

was measured as the densitometry of enolase activity divided by the amount of c-SRC per lane,

and normalized to the DMSO control treatment.

c-SRC Adenovirus Gene Transduction: Recombinant Ad5/F35 adenovirus vectors were

created by Daniel Jung. These bicistronic vectors contained enhanced yellow fluorescent protein

97

(EYFP), and expressed wild type human c-SRC (WT), a dominant negative, kinase inactive c-

SRC (DN) or no c-SRC as a control (empty vector; EV) [190]. Cells were titrated with virus to

determine the highest multiplicity of infection that permitted 70-80% gene transduction, while

minimizing cell death 48 hours post-infection. EYFP detection and viability staining (7-AAD,

which stains both apoptotic cells and necrotic bodies, BD Sciences, Franklin Lakes, USA) were

performed on fixed cells using FACS (see flow cytometry and data acquisition).

siRNA Knockdown: Pools of four siRNA duplexes, specific for human c-SRC (cat # M-

003175-03) or PTK2B (cat # M-003164-02) mRNA, and a control pool of non-targeting siRNA

(cat # D-001206-13-05) were ordered from Dharmacon RNAi technologies (siGENOME

SMART Pool, Thermo Scientific, Lafayette, USA). In Jurkat E6-1 cells, siRNA knockdown

was achieved using the siRNA cationic lipid transfection reagent GeneSilencer® (Genlantis, San

Diego, USA), following the manufacturer’s instructions. Two μL of siGuard RNase inhibitor®

(Genlantis) was added to each 1 mL reaction to prevent RNAse degradation of the RNA

duplexes. Upon siRNA titration, 900 nM was determined to give the optimal level of protein

knockdown for both c-SRC and PTK2B, via Western blot densitometry at 48 hours.

HIV-1 Infection and Luciferase Assay: 1 x 106 T-cells pre-treated with drug, adenovector or

siRNA were infected with 1.4 ng of VSV-G/HIV-1 or 7.5 ng of HXB2 at 37°C in 2 mL of

growth media in 12-well plates for 2 days. Cells were counted, while others were lysed with the

Luciferase Assay System (Promega, Madison, USA) or kept for Western blots.

Antibodies/Immunoblots: Jurkat C cells were serum-starved and then immediately following

15 minutes of HIV-IIIB infection, were washed in PBS and lysed with RIPA buffer. Monoclonal

anti-phosphotyrosine (anti-ptyr) clone 4G10 (Millipore) and protein G-Sepharose beads (Santa

Cruz Biotechnology, Dallas, USA) were used to immunopreciptate phosphoproteins from 4 x 106

Jurkat C cell lysates.

Following VSV-G/HIV-1 or HXB2 infection, 5-10 x 105 T-cells were washed in PBS and lysed

with RIPA buffer. After boiling samples, proteins were resolved on an 8%, reducing SDS-

PAGE gel, and transferred to an Immobilon-P membrane for Western Blot detection. The blots

98

were blocked with 5% skimmed-milk powder and 0.5% Nonidet P-40 (BioShop, Burlington,

Canada). Primary antibodies diluted in 5% milk and 0.5% Nonidet P-40, anti-β-actin (Sigma),

anti-α-tubulin (Sigma) anti-PTK2B (Transduction Laboratories) and anti-c-SRC GD11

(Millipore) were used to probe the blots. After washing, secondary goat anti-mouse HRP

antibody (Bio-Rad, Mississauga, Canada) was added, followed by enhanced chemiluminescent

(ECL) detection (GE Healthcare, Buckinghamshire, UK). PTK2B and c-SRC protein levels were

determined by comparing their densitometry to normalizers β-actin or α-tubulin with the

Molecular Imager GelDoc XR+ Imagin System (Bio-Rad).

DNA isolation and quantitative PCR: Genomic and viral cDNA was isolated from 5 x 105

Jurkat E6-1 cells, pre-treated by siRNA transfection and infected with HXB2 virus for 1 or 12

hours, using the QIAamp DNA Blood Mini Kit (Qiagen, Toronto, Canada). Samples were

heated at 56°C for 2 hours in QIAamp DNA Blood Mini Kit buffer AL to deactivate viable viral

particles.

The primer sets used to detect human or viral DNA (synthesized by the Center for Applied

Genomics, The Hospital for Sick Children, Toronto, Canada) were as follows [159, 542, 543]: β-

globin forward, 5′-CCCTTGGACCCAGAGGTTCT -3′; β-globin reverse, 5′-CGAGCACTTTCT

TGCCATGA-3′; early reverse transcripts (early RT) forward, 5′-GTAACTAGAGATCCCTCAG

ACCCTTTTAG-3′; early RT reverse, 5′- TAGCAGTGGCGCCCGA-3′; late reverse transcripts

(late RT) forward, 5′-CCGTCTGTTGTGTGACTCTGG-3′; late RT reverse, 5′-GAGTCCTGCG

TCGAGAGATCT-3′; genomic Alu forward, 5′-GCCTCCCAAAGTGCTGGGATTACAG-3′;

HIV-1 gag reverse, 5′-GCTCTCGCACCCATCTCTCTCC-3′; HIV-1 LTR forward, 5′-GCCTCA

ATAAAGCTTGCCTTGA-3′; HIV-1 LTR reverse, 5′-TCCACACTGACTAAAAGGGTCTGA-

3′. For each qPCR run, 50-100 ng template DNA (with the exception of the no-template control,

NTC) was added to PCR tubes containing: 12.5 μL 2x SYBR Green PCR Master Mix (Applied

Biosystems), 300-900 nM of each forward and reverse primers, and PCR grade H2O (Roche

Diagnostics, Indianapolis, USA) up to a final volume of 25 μL. PCR cycling parameters were as

follows: initial denaturation at 95°C for 10 min; 40-50 cycles of amplification of 95°C for 15

sec, 58°C for 30 sec (β-globin or late RT, 54°C for early RT) and 60°C for 30 sec. To measure

integrated virus by Alu-gag PCR [543], tubes were first pre-amplified with 100 nM Alu forward

99

and 600 nM gag reverse primers, with 2x SYBR Green PCR Master Mix as above, for 10 cycles

of 93°C for 30 sec, 50°C for 60 sec, and 60°C for 100 sec. Nested PCR was performed on these

samples by adding 300 nM each of HIV-1 LTR forward and reverse primers, with 2x SYBR

Green PCR Master Mix, for 40 more cycles using the above parameters. Duplicate reactions

were analyzed using the Rotor-Gene RG-3000 thermocycler (Corbett Research, Montreal,

Canada). c-SRC and PTK2B siRNA-treated groups were compared to the non-targeting siRNA-

treated group by the 2-ΔΔCT

comparative CT method [544].

Flow Cytometry and data acquisition: Cell surface expression of CD4 and CXCR4 were

determined on 2.5 x 105 Jurkat E6-1 cells per FACS tube, 48 hours after siRNA knockdown.

Cell surface staining was performed as previously described [545]. In brief, cells were stained

with 5 μL of FITC mouse anti-human CD4, 5 μL of PE mouse anti-human CXCR4 and 2.5 μL of

7-AAD, in 100 μL of FACS buffer (PBS + 2.5% FBS). Isotype control antibodies were FITC-

conjugated mouse IgG1κ and PE-conjugated mouse IgG2aκ. All of these antibodies were

purchased from BD Pharmingen, Mississauga, Canada. Data on 20,000 events per tube was

collected using Becton Dickenson FACSCalibur (BD Calibrite; BD Biosciences). Signals were

acquired for the forward scatter (FSC), side scatter (SSC), the green (FITC, EYFP), yellow (PE)

and red (7-AAD) channels.

Reverse Transcriptase Activity Assay: Quantification of reverse transcriptase activity from

infected cell lysates was performed using the SYBR Green I qPCR-based product-enhanced RT

(PERT) assay [546]. Briefly, 5 x 105 JE6-1 cells were infected with HXB2 virus for 1 hr,

washed twice with PBS, lysed, diluted 10-fold with 10x sample dilution buffer, and given 1 μg

of MS2 RNA template (Roche Diagnostics) to reverse transcribe in vitro. The cDNA product

was measured by qPCR, and a standard curve of recombinant HIV-1 RT (Calbiochem) was used

to assign enzyme activity units to reverse transcriptase from the infected cell lysate samples.

Statistical Analysis: Means were compared using a two-tailed, unpaired Student’s t test and

corrected for multiple comparisons when more than two means were considered in an

experiment, to minimize the probability of type 1 errors. FACS data shows the Median

Fluorescent Intensity (MFI) and percent positive cells collected with CellQuest software (BD

100

Biosciences) and analyzed by FlowJo version X software. Comparisons of histogram EYFP

expression was performed with the two sample Kolmogorov-Smirnov test. For all figures, an

asterisk (*) denotes a p value < 0.05, two asterisks (**) denotes a p value < 0.01 and three

asterisks (***) denotes a p value < 0.005. Error bars shown are the standard error around the

mean (SEM).

101

2.4 Results

Tyrosine kinases become activated within minutes of HIV-1IIIB infection

To confirm previous reports that non-receptor kinases become activated within minutes of HIV-1

infection, we infected serum-starved Jurkat C T-cells with HIV-1IIIB, HXB2 (X4 strains), or with

VSV-G pseudoenveloped HIV-1 virus. We then immunoprecipitated protein substrates from T-

cell lysates with anti-ptyr clone 4G10 (Fig. 2.1). Probing the blot of HIV-1IIIB infected cell lysate

with anti-ptyr revealed a robust phosphorylation of substrates in the 55-130 kDa range, and one

low molecular band at ~40-42 kDa (Fig. 2.1A). Re-probing the Western blot for PTK2B and c-

SRC (Fig. 2.1B), demonstrated phosphorylation of these kinases within 15 minutes of HIV-1

infection, indicative of their activation and recruitment during early HIV-1 entry events.

102

Fig. 2.1: Western blot of phosphorylated proteins from serum-starved T-cells shortly after

HIV-1 infection. 4 x 106 Jurkat C T-cells were infected with 22.8 ng of VSV-G/HIV-1, HXB2

or HIV-1IIIB for 15 minutes, washed in PBS and lysed with RIPA buffer. Phosphoproteins were

than immunoprecipitated using monoclonal anti-phosphotyrosine (anti-ptyr) clone 4G10, and run

on a 10% reducing SDS-PAGE gel. Lane one is Jurkat C cell lysate, lane two is anti-ptyr

antibody, and lane three is immunoprecipitated infected Jurkat C cell lysate. A Western blot

transfer was performed and the blot was probed for total phosphoproteins with anti-ptyr clone

4G10 (Panel A), anti-PTK2B or anti-c-SRC GD11. Panel B shows the densitometry of tyrosine

phosphorylated PTK2B and c-SRC in a single experiment.

103

SU6656 consistently inhibited c-SRC activity, and increased VSV-G/HIV-1 infection

To directly implicate a role for c-SRC activity during early HIV-1 infection, we used well known

c-SRC inhibitors (PP1, PP2 and SU6656) [184] to inhibit c-SRC activity prior to HIV-1

infection. For these experiments a replication deficient, pseudotyped nef-deficient VSV-G/HIV-

1 virus carrying a luciferase reporter gene was used, so as not to produce viral protein products

which are known to directly bind and activate the SRC family of kinases, such as viral protein

Nef [164]. After pre-treating Jurkat E6-1 cells with 10 μM of drug for 1 hour, only the SU6656

treated cells showed a significant increase in VSV-G/HIV-1 infection after two days, compared

with DMSO control (Fig. 2.2A). Cell growth was not adversely affected during this experiment

(Figure 2.3A). The non-specific effects of SU6656 differ from PP1 and PP2 [184], which is why

we decided to move forward using SU6656 as our c-SRC inhibitor. Our in-vitro kinase assay

showed that SU6656 reduces c-SRC activity by 50% at 10 μM (Fig. 2.2B). We repeated our

VSV-G/HIV-1 infection experiment at two different SU6656 drug concentrations (Fig. 2.2C) and

in another Jurkat T-cell line (Fig. 2.2D) to confirm our finding that c-SRC drug inhibition leads

to increased VSV-G/HIV-1 infection in T-cell lines.

104

Fig. 2.2: Jurkat T-cell lines inhibited with SRC-family kinase inhibitors. Panel A: 2 x 105

Jurkat E6-1 cells were pre-treated with 10 μM of DMSO (control), PP1, PP2 or SU6656 for 1 hr,

washed in PBS, then infected with 1.4 ng of VSV-G/HIV-1 for two days. Cells were then

counted, while other cells were lysed to measure luciferase activity (RLU) to quantify viral

infection. This experiment was repeated 3 times. Panel B: After treating 2 x 105 Jurkat C cells

with 5 or 10 μM SU6656 for 1 hr in a single experiment, cells were lysed, c-SRC protein was

immunoprecipitated, and in-vitro kinase activity was determined by the phosphorylation of

enolase substrate with γ32

P-ATP. Panel C and D: 2 x 105 Jurkat E6-1 cells (C) or Jurkat C cells

(D) were pre-treated with 10 or 20 μM of SU6656, and then infected with 1.4 ng of VSV-

G/HIV-1 for two days. Luciferase activity was then measured. Standard error bars are of

triplicate measurements.

105

Fig. 2.3: Cell survival was not significantly different between c-SRC drug or adenovirus

vector treatments. Panel A: 2 x 105 Jurkat E6-1 cells were pre-treated with 10 μM of DMSO

(control), PP1, PP2 or SU6656 for 1 hr, washed in PBS, and then grown for two days in RPMI.

Cells were then counted using trypan blue stain exclusion. Panel B: T-cell lines were

transduced with adenovectors expressing c-SRC mutants. Adenovirus vectors (EV = empty

vector, WT c-SRC = Wild Type c-SRC, DN c-SRC = Dominant Negative c-SRC) were

administered to Jurkat E6-1, Hut 78 and Kit 225 cells at an MOI of 750 for 48 hr. The percent of

live cells (7-AAD -ve) was then measured using FACS in a single experiment.

A

B

106

Adenovector expressing dominant negative c-SRC increased VSV-G/HIV-1 infection in

CD4+ T-cell lines

As with most kinase inhibitors, we cannot ignore that SU6656 inhibits related SRC family

members, such as LCK and FYN, in conjunction with c-SRC. Thus, we used adenovirus gene

transduction to alter the pool of functional c-SRC in three T-cell lines, prior to VSV-G/HIV-1

infection. Jurkat E6-1, Hut 78 and Kit 225 T-cells were transduced with adenoviruses containing

an Empty Vector control (EV) Wild Type (WT) c-SRC or a Dominant Negative (DN) c-SRC

that can bind but not phosphorylate substrate (Figure 2.4). Administering adenovector

expressing WT c-SRC or DN c-SRC did not alter cell survival when compared to EV, within

each cell type (Figure 2.3B). Once we optimized multiplicity of infection (MOI) to reduce cell

death and maintain high gene transduction, we achieved 62-84% EYFP expression in Jurkat E6-1

and Hut 78 cells after 2 days (Fig. 2.4B-C). Kit 225 cells were less transducible (36-47%, Fig.

2.4D), but showed higher overall survival. No statistical difference was found between EV, WT

c-SRC or DN c-SRC EYFP expression, by the Kolmogorov-Smirnov test.

Viral infection was normalized to the total cells in each well after two days of VSV-G/HIV-1

infection. At this time point, no significant difference in VSV-G/HIV-1 infectivity was found

between EV and WT c-SRC treatments (Fig. 2.4E-G). However, administering the DN c-SRC

adenovector caused a significant increase in VSV-G/HIV-1 infection in two of the three T-cell

lines tested. This finding complements our previous c-SRC drug experiments. Competing

endogenous c-SRC enzyme with the dominant negative mutant increases the integration or

transcription of VSV-G/HIV-1.

107

Fig. 2.4: T-cell lines were transduced with adenovectors expressing c-SRC mutants.

Adenovirus vectors (EV = empty vector, WT c-SRC = Wild Type c-SRC, DN c-SRC =

Dominant Negative c-SRC) were administered to Jurkat E6-1, Hut 78 and Kit 225 cells at an

MOI of 750 for 48 hr. FACS gating was performed on live cells that were 7-AAD negative

(Panel A). On this cell subset, EYFP was measured to determine gene transduction efficiency

(Panels B-D). Median fluorescent intensity (MFI) and % positive cells are shown. After two

days of adenovirus gene transduction, 2 x 105 cells were infected with 1.4 ng VSV-G/HIV-1, and

luciferase activity measured after an additional two days (Panels E-G). Standard error bars are

of triplicate measurements.

108

Both c-SRC and PTK2B knockdown caused an increase in either VSV-G/HIV-1 or HXB2

infection.

To assess whether the upstream binding partner PTK2B mediates the effects of c-SRC during

HIV-1 infection, we used siRNA to knockdown mRNA expression of either kinase in Jurkat E6-

1 cells. Relative to a non-targeting siRNA control, cell proliferation was not affected by PTK2B

or c-SRC knockdown (Figure 2.5A). T-cells are a challenging non-adherent T-cell line to

transfect for siRNA knockdown, hence we optimized the siRNA concentration delivered and the

time-point to assess protein knockdown by Western blot (Fig. 2.5B-G). Qualitatively, we found

900 nM of siRNA transfected by lipofection could reliably knockdown either c-SRC or PTK2B

by 40-50%, 48 hours post-transfection (Fig. 2.6A-D).

At this time point, we then infected the Jurkat E6-1 cells with VSV-G/HIV-1 and measured

infection two days later. Interestingly, both c-SRC and PTK2B knockdown in Jurkat E6-1 cells

lead to an increase in luciferase activity upon infection with the VSV-G/HIV-1 reporter virus,

when compared with the non-targeting siRNA control group (Fig. 2.6E). This implicates both

kinases as having important catalytic activity during early HIV-1 infection of T-cells. To

determine whether this phenomenon can be attributed to the differences in vesicular stomatitis

entry into a host T-cell (clathrin-dependent endocytosis) [547] and that of HIV-1 (receptor-

mediated internalization), we performed the siRNA knockdown experiment once more with an

HXB2 (X4) luciferase reporter virus that is also replication-deficient. Similar to the VSV-

G/HIV-1 infection, when either c-SRC or PTK2B protein expression is reduced by siRNA

knockdown, we saw a concomitant increase in HXB2 viral infection after two days (Fig. 2.6F).

This suggests that PTK2B and c-SRC may play similar roles in the early HIV-1 entry events of

both viruses downstream of HIV-1 receptor binding and fusion.

109

Fig. 2.5: siRNA titration and time-course optimization in Jurkat E6-1 cells. Panel A: Jurkat

E6-1 cell survival two days post-transfection of non-targeting, c-SRC or PTK2B siRNA, as

measured by trypan blue staining. Panels B-E: Two days post-lipofection, 5 x 105 cells were

lysed with RIPA to perform an 8% SDS-PAGE followed by a Western blot. Blots were probed

with anti-c-SRC (B and C), anti-PTK2B (D and E) and anti-α-tubulin. Quantification of PTK2B

and c-SRC protein was relative to α-tubulin control. Panels F and G: Time-course for optimal

c-SRC knockdown post-lipofection. All of these were single experiments.

B

C

D

F

G E

A

110

Fig. 2.6: siRNA knockdown in Jurkat E6-1 cells infected with either VSV-G/HIV-1 or

HXB2 virus. Panels A and B: Two days post-transfection with non-targeting, c-SRC or

PTK2B siRNA, 5 x 105 cells were lysed with RIPA to perform an 8% SDS-PAGE followed by a

Western blot. Blots were probed with anti-PTK2B, anti-c-SRC or anti-α-tubulin. Quantification

of PTK2B and c-SRC protein was relative to α-tubulin control, and normalized to the non-

targeting siRNA treatment. Original Western blots are shown in Panels C and D and are

representative of three independent experiments. Two days after siRNA transfection, 2 x 105

cells were plated and infected with 1.4 ng of VSV-G/HIV-1 (Panel E) or 7.5 ng of HXB2 virus

(Panel F). Luciferase activity was measured in cell lysates 48 hr thereafter.

111

c-SRC and PTK2B siRNA knockdown increased the reverse transcription of HXB2 cDNA

To assess at which early stage c-SRC or PTK2B were having their effect, we measured the

production of HIV-1 early reverse transcripts (early RT) late reverse transcripts (late RT), as well

as integrated virus, shortly after HXB2 infection by real-time qPCR (Fig. 2.7A). One hour post-

infection, both c-SRC and PTK2B siRNA knockdown led to a 4-10 fold increase in early and

late reverse transcripts, relative to non-targeting siRNA treated cells. Knocking down c-SRC or

PTK2B also led to a 3-4 fold increase in integrated virus measured after 12 hours of infection.

We detected no increase in CD4 or CXCR4 cell surface expression after c-SRC or PTK2B

knockdown (Fig. 2.7B), suggesting the increase in HIV-1 early RT, late RT and integrated virus

is not due to increased receptor binding upon entry. Measuring reverse transcriptase activity 1

hour post-infection revealed increased activity from lysates of Jurkat T-cells administered c-SRC

or PTK2B siRNA (Fig. 2.7C), suggesting that they act, at least in part, at the level of HIV-1

reverse transcription.

112

Fig. 2.7: qPCR of HXB2 cDNA extracted from Jurkat E6-1 following siRNA knockdown.

Shortly after infection, early reverse transcripts (early RT) at 1 hr, late reverse transcripts (late

RT) at 1 hr, and integrated virus at 12 hr post-infection were measured relative to β-globin

expression (Panel A). FACS analysis of CD4 and CXCR4 expression on Jurkat E6-1 cells

following 48 hr of siRNA knockdown, gating on the 7-AAD negative population (Panel B).

Reverse transcriptase activity was measured from lysates of 5 x 105

cells after 1 hr of HXB2

infection (Panel C). Standard error bars are of five replicate measurements in A and C.

113

2.5 Discussion

The present study demonstrates that reducing c-SRC enzyme activity, outcompeting endogenous

c-SRC with a dominant negative SRC mutant, or reducing c-SRC protein levels with siRNA

knockdown caused an increase in VSV-G/HIV-1 infection in various T-cell lines. We first

demonstrated that c-SRC becomes activated within 15 minutes of HIV-1IIIB infection, which

agrees with our previous findings [214]. We also found that PTK2B becomes phosphorylated in

the same 15 minute time span, faster than Seror et al. reported using HIV-1NDK virus [345].

Reducing c-SRC activity with SU6656 consistently caused an increase in VSV-G/HIV-1

infection in Jurkat T-cells. Initially, this finding appears paradoxical considering that CD4+ T-

lymphocytes pre-treated with PP2 were previously reported to show a decrease in p24 levels six

days after NL4-3balenv or JR-CSF (R5) infection [264]. This discrepancy may be because our

viral read-out measured transcription of luciferase to detect successful viral integration, instead

of p24 to monitor viral production and egress. The initial activation of non-receptor tyrosine

kinases is rapid [214], and kinases that play a role during early entry may have different roles

during viral egress and p24 release. Thus inhibiting c-SRC activity may promote early entry of

HIV-1, as we have found, and yet reduce viral p24 production at later stages of the viral

replication cycle. This difference in function could perhaps be attributed to the downstream

substrate, the 68 kDa SRC-associated protein in mitosis (Sam 68), which is known to assist in the

nuclear export and translation of HIV-1 RNA [317].

We also observed that the addition of adenovector producing dominant negative c-SRC in three

T-cell lines caused an increase in VSV-G/HIV-1 infection, yet adding adenovector expressing

wild type c-SRC did not reduce infection. Perhaps this can be attributed to the dynamic

regulation of c-SRC by cellular phosphatases (SHP-1, CD45) and kinases (CSK), explaining why

the addition of wild type c-SRC could not reduce viral infection. Nonetheless, the ability of

dominant negative c-SRC to compete with endogenous c-SRC for substrates and cause an

increase in VSV-G/HIV-1 infection agrees well with our drug experiments. Whether c-SRC

directly phosphorylates HIV-1 proteins, or triggers signaling pathways that hinder viral entry and

integration, warrants further investigation.

114

In terms of our siRNA knockdown findings, both c-SRC and PTK2B knockdown caused an

increase in VSV-G/HIV-1 infection in Jurkat T-cells. This agrees well with the previous finding

that activated PTK2B links G protein-coupled receptors to downstream signaling by complexing

with c-SRC and subsequently causing c-SRC activation [197]. It is interesting to note that while

VSV-G/HIV-1 enters cells via clathrin-dependent endocytosis [547], using a virus with HXB2

(X4) envelope gave a similar result, suggesting both kinases play important roles in HIV-1

infection regardless of initial entry route. We also found increased reverse transcriptase activity,

early reverse transcripts, late reverse transcripts and integrated virus after c-SRC or PTK2B

siRNA knockdown. PTK2B, being upstream of c-SRC, had a stronger effect on viral DNA

produced in cells. It will be of interest to determine the mechanism by which these kinases affect

early infection, perhaps stabilizing the reverse transcriptase complex or pre-integration complex

through phosphorylation or allosteric interactions.

Our research is the first study to explore the impact of c-SRC and PTK2B activation on early

HIV-1 infection in CD4+ T-cell lines. Discovering novel mechanisms in the entry of HIV-1 and

their impact on T-cell signal transduction may lead to targeting these pathways with drugs and

improving the treatment options available to people with HIV-1. Tyrosine kinase inhibitors with

low toxicity profiles exist for cancer therapies, and could be repositioned as viable options for

treating HIV-1 infection. For instance cyclin-dependent kinase inhibitors, such as r-roscovitine

and alsterpaullone, are showing promising anti-HIV-1 properties in PBMCs by preventing viral

Tat transactivation [548]. We also know that tyrosine kinase inhibitors can be safe in HIV-1

patients, as a case study of patients with AIDS-related Kaposi Sarcoma given the ABL kinase

inhibitor imatinib showed no increase in viral plasma load [176]. Our results show increased

HIV-1 early reverse transcripts, late reverse transcripts and integration when c-SRC or PTK2B

are inhibited, which may preclude the use of SRC inhibitors during early HIV-1 infection.

However, the potential use of c-SRC kinase inhibitors to study the stability of the reverse

transcriptase complex and pre-integration complex warrants further investigation.

115

Chapter 3: c-SRC Protein Tyrosine Kinase Regulates

Early HIV-1 Infection Post-Entry

Role of author McCarthy S.D.: Performed all the experiments for Figures 3.1-3.10 and

illustrated Figure 3.11. I analyzed all the data, wrote the manuscript draft, and performed

experiments to address reviewer’s comments.

A version of this chapter was published in the Journal AIDS:

McCarthy, S.D., Sakac, D., Neschadim, A., and Branch, D.R. 2016. c-SRC protein tyrosine

kinase regulates early HIV-1 infection post-entry. AIDS, 30(6): 849-58.

116

3.1 Abstract

Objective: We investigated whether HIV-1 inhibition by SRC-family kinase inhibitors is

through the non-receptor tyrosine kinase phosphoprotein pp60c-SRC

(c-SRC) and its binding

partner, protein tyrosine kinase 2 beta (PTK2B).

Design: CD4+ T-lymphocytes were infected with R5 (JR-FL) or X4 (HXB2) HIV-1. SRC-

family kinase inhibitors or targeted siRNA knockdown of c-SRC and PTK2B were used and

various stages of the early HIV-1 lifecycle were examined.

Methods: Four SRC-family kinase inhibitors or targeted siRNA knockdown were used to reduce

c-SRC or PTK2B protein expression. Activated CD4+ T-lymphocytes were infected with

recombinant, nef-deficient, or replication-competent infectious viruses. Knockdown experiments

examined multiple stages of early infection by monitoring: luciferase activity, expression of host

surface receptors, reverse transcriptase activity, p24 levels and qPCR of reverse transcripts,

integrated HIV-1 and 2-LTR circles.

Results: All SRC-family kinase inhibitors inhibited R5 and X4 HIV-1 infection. Neither c-

SRC nor PTK2B siRNA knockdown had an effect on cell surface receptors (CD4, CXCR4,

CCR5) nor on reverse transcriptase activity. However, using JR-FL both decreased luciferase

activity while increasing late reverse transcripts (16-fold) and 2-LTR circles (8-fold) while also

decreasing viral integration (4-fold). With HXB2, c-SRC but not PTK2B siRNA knockdown

produced similar results.

Conclusions: Our results suggest c-SRC tyrosine kinase is a major regulator of HIV-1 infection,

participating in multiple stages of infection post-viral entry: Reduced proviral integration with

increased 2-LTR circles is reminiscent of integrase inhibitors used in combination antiretroviral

therapies. Decreasing c-SRC expression and/or activity provides a new target for antiviral

intervention and the potential for repurposing existing FDA-approved kinase inhibitors.

117

3.2 Introduction

To overcome HIV-1 drug resistance, targeting host T-cell factors critical to HIV-1 infection has

become a promising field of research [146]. Therapeutics designed to target host proteins are

being explored in conjunction with combination antiretroviral therapy (cART) to reduce the size

of the initial HIV-1 reservoir [549] or reactivate then purge HIV-1 in the cells of infected people

[550]. For example, some drugs disrupt allosteric interactions between viral integrase and host

proteins LEDGF/p75 [551], and other therapeutics can inhibit histone deacetylases to reactivate

provirus in latently infected cells [552]. However, inhibition of tyrosine kinases activated during

early HIV-1 infection remains an underexplored area of antiretroviral drug discovery. From

initial binding of viral glycoprotein 120 (gp120) to the T-lymphocyte receptor CD4 and

chemokine co-receptor CXCR4 (X4 viruses) or CCR5 (R5 viruses), tyrosine kinases are induced

that promote viral infection [147]. Moreover, non-receptor tyrosine kinases play critical roles

during the early stages of the HIV-1 lifecycle in T-cells (reviewed in [146]). Tyrosine

phosphorylation signaling is necessary for viral entry [135], actin remodeling [539] and

translocation of the viral pre-integration complex (PIC) into the nucleus [154, 158]. In

particular, HIV-1 infected CD4+ T-cells show striking changes in the activity of the SRC-family

of tyrosine kinases, which become activated within minutes of HIV-1 infection [214]. Previous

work suggests that dasatinib, a dual SRC/ABL ATP competitive inhibitor, approved for the

treatment of imatinib-resistant chronic myeloid leukemia [553], effectively inhibits HIV-1

production in activated CD4+ T-cells isolated from chronically infected HIV-1 patients [554].

However, a specific role for c-SRC at earlier time points, post initial infection up through

integration, has not been examined.

When HIV-1 initially attaches to the host T-cell membrane through its interaction with cell-

surface CD4, viral gp120 then binds the G protein-coupled receptor CXCR4/CCR5, inducing the

autophosphorylation of protein tyrosine kinase 2 beta (PTK2B, Pyk2 or FAK2) within the cell

[147]. PTK2B plays a key role in focal adhesions at the cell periphery and in cytoskeleton

remodeling [340]. Phosphorylation of PTK2B at Tyr402 can then recruit c-SRC (SRC, pp60c-

SRC) to its SRC-homology-2 (SH2) binding domain, permitting c-SRC to autophosphorylate at

118

Tyr419, activating c-SRC [197, 322]. Both of these kinases become phosphorylated within

minutes of HIV-1 infection [147, 214].

Previously, we have reported that inhibiting or reducing PTK2B or c-SRC in various T-cell lines

increased HIV-1 reverse transcription and integrated provirus [555]. However, another study has

shown that PTK2B shRNA knockdown in the T CEM cell line reduced viral p24 production after

six days of HIV-1NDK infection [345]. In addition, Gilbert et al. have demonstrated that

inhibiting c-SRC with the kinase inhibitor PP2 decreased p24 production in primary CD4+ T-

cells after six days of infection [264]. The discrepancy of PTK2B and c-SRC inhibition that

promotes early HIV-1 entry within hours, yet restricts virion maturation and p24 release within

days, remains unresolved. One possibility is that PTK2B and c-SRC function differently during

early HIV-1 entry in activated primary CD4+ T-lymphocytes than in immortalized T-cell lines.

To test this possibility, we hypothesized that inhibiting the non-receptor tyrosine kinases PTK2B

and c-SRC will restrict HIV-1 early entry in activated primary CD4+ T-cells.

We demonstrate that reducing c-SRC expression with siRNA restricted the infection of X4-tropic

HXB2 or R5-tropic JR-FL, Ba-L or KNH1207, within 12 hrs of infection. Interestingly, PTK2B

siRNA knockdown only reduced early infection of R5-tropic viruses, suggesting different

dependence on downstream signaling contingent on the co-receptor engaged during virus

binding and fusion. Our findings not only provide the rationale for exploring the use of drugs to

inhibit these two kinases as novel means to inhibit HIV-1 replication, but they may also suggest

a unique method for arresting HIV-1 at the stage of pre-integration complex (PIC) formation

and/or integration.

119

3.3 Materials and Methods

Cell Isolation and Culture Conditions: Peripheral blood mononuclear cells (PBMCs) were

isolated from the whole blood of healthy human donors (Canadian Blood Services Research

Ethics Board Committee Protocol Reference #2005-003) by the Ficoll gradient method,

according to manufacturer’s instructions (GE healthcare, Buckinghamshire, UK). Informed

written consent was obtained from all subjects. CD4+ T-cells were then isolated from PBMCs

using the EasySep negative selection human CD4+ T-cell enrichment kit, following the protocol

by StemCell Technologies (Vancouver, Canada). For all experiments, isolated CD4+ T-cells

were grown in complete RPMI-1640 media (Sigma-Aldrich, Oakville, Canada) containing 10%

fetal bovine serum, 1% penicillin, 1% streptomycin and 0.1% gentamycin. Human embryonic

kidney cells (HEK 293T, ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM,

Gibco, Burlington, Canada) with the same proportion of FBS and antibiotics as above. Cell

cultures were incubated at 37°C in 5% CO2 atmosphere. CD4+ T-cells were activated by the

addition of the following: 10 μg/mL anti-CD3 (UCHT-1, Antibody Core Facility, Sunnybrook

Research Institute, Toronto, Canada), 2 μg/mL anti-CD28 (10R-CD28bHU, Fitzgerald Industries

International, Acton, USA) and 10 units/mL recombinant human Interleukin-2 (IL-2, Sigma-

Aldrich).

Virus Production: We produced recombinant, replication deficient JR-FL enveloped (R5) or

HXB2 enveloped (X4) HIV-1 containing a luciferase gene, as described previously [555].

Briefly, 2.5 x 106 HEK 293T cells were plated with 10 mL of DMEM 24 hours prior to

transfection. We co-transfected 10 μg of HIV-1 NL4-3luc (luciferase gene inserted into viral nef

gene and env deficient; a kind gift from Dr. Veneet KewalRamani, New York Medical

University, NY) with 10 μg of JR-FL env plasmid (encodes the R5-tropic JR-FL glycoproteins; a

generous gift from Dr. M. Tremblay, Quebec City, PQ) into each 10 cm plate of HEK 293T cells

with the CalPhosTM

Mammalian Transfection Kit (Clontech, Mountain View, USA), following

the manufacturer’s instructions. This occurred in a biosafety level 3 (BSL3) lab. The same was

performed for the HXB2 plasmid (HIV-1 NL4-3luc lacking nef and containing HXB2 (X4) env;

another generous gift from Dr. M. Tremblay) at 15 μg per plate. DMEM media was replaced

120

after 24 hours and viral supernatant was collected on days 2 and 3. The supernatant was

syringed through 0.45 μm pore filters (Millipore, Billerica, USA) , then concentrated by

underlaying with a 20% sucrose gradient and centrifuging at 16,000 x g for 90 minutes at 4 °C.

Virus pellet were resuspended in TNE buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA and 100

mM NaCl) and stored in -80°C aliquots. p24 levels were measured by ELISA (ZeptoMetrix,

Buffalo, USA). Replication competent IIIB (formerly HTLV-III B/H9), Ba-L (Reference ID:

85US_Ba-L) and KNH1207 (Reference ID: 00KE_KNH1207) viruses were acquired from the

NIH AIDS Research & Reference Reagent Program’s International panel of 60 HIV-1 isolates

(Germantown, USA). All viruses underwent one freeze-thaw cycle prior to infection studies.

HIV-1 Infection and Luciferase Activity: 2 x 105 CD4

+ T-cells pretreated with siRNA or drugs

were infected with 1.0 ng of HXB2, 26 ng of JR-FL, 1.1 ng IIIB, 43 ng of Ba-L or 16 ng of

KNH1207 at 37°C in 200 μL of clear growth media including activators in 96-well plates for 2

days. Cells were counted, while others were lysed with the Luciferase Assay System (Promega,

Madison, USA). Other CD4+ T-cells were infected with replication competent IIIB (X4), Ba-L

(R5) or KNH1207 (R5), and integrated copies of provirus were quantified by qPCR after 12 hrs

of infection.

HIV-1 p24 ELISA: 2 x 105 CD4

+ T-cells pretreated with siRNA were infected with IIIB or Ba-

L for 1 hr at 37°C, washed in PBS three times, and incubated in 200 μL complete RPMI media

supplemented with activators. Cell-free supernatant (180 μL) was collected on day 3 and assayed

for p24gag

antigen following the manufacturer’s instructions (ZeptoMetrix, Franklin, USA).

Drug Experiments: Enriched CD4+ T-cells were administered potent small molecule c-SRC

inhibitors. Cells were given complete media containing activators, and one of the four inhibitors:

dasatinib (BMS-354825), saracatinib (AZD0530), KX2-391 (Selleckchem, Houston, USA) or

SRC Inhibitor-1 (Sigma-Aldrich). DMSO solvent served as a negative control. Twenty-four

hours thereafter, cells were prepared for HIV-1 infection or FACS viability staining (Annexin V-

FITC Apoptosis Detection Kit I, BD Pharmingen, Mississauga, Canada) to measure apoptosis

and/or necrosis. In other experiments, enriched CD4+ T-cells were given 20 μM of raltegravir 24

hours prior to HIV-1 infection.

121

Flow Cytometry and Data Acquisition: All antibodies were purchased from BD Pharmingen.

Cell surface expression of CD3 and CD4 were determined on 1 x 106 CD4

+ T-cells after using

the EasySep negative selection human CD4+ T-cell enrichment kit (StemCell). In other

experiments, cell surface expression of CD4, CXCR4 and CCR5 were determined on 2 x 105

enriched CD4+ T-cells per tube, 48 hours after siRNA knockdown. In brief, cells were stained

with 5 μL of PE mouse anti-human CD3, 5 μL of APC mouse anti-human CD4, 5 μL of FITC

mouse anti-human CD4, 5 μL of PE mouse anti-human CXCR4, or 10 μL of Alexa Fluor®647

rat anti-human CCR5 in a 100 μL of FACS buffer (PBS + 2.5% FBS). Isotype control

antibodies were FITC-conjugated mouse IgG1κ, PE-conjugated mouse IgG1κ or IgG2aκ, APC-

conjugated mouse IgG1κ and Alexa Fluor®647-conjugated rat IgG2a. Data on 20,000 events per

sample was collected using the Becton Dickenson FACSCalibur flow cytometer (BD

Biosciences, Mississauga, Canada).

Antibodies and Western Blot: Activated CD4+ T-cells (1 x 10

6) infected with HXB2 or JR-FL

virus for 1 hr were washed in PBS and lysed with radioimmunoprecipitation assay (RIPA) lysis

buffer (50 mM HEPES, pH 7.3, 1% Nonidet P-40, 0.1% SDS, 0.1% sodium deoxycholate, 150

mM NaCl, 1 mM Na3VO4, 50 M ZnCl2, 2 mM EDTA and 2 mM PMSF). In other co-

immunoprecipitation experiments, 3x106 activated CD4

+ T-cells were infected with HXB2 or

JR-FL for 1 hr, lysed, and rotated at 4°C overnight with protein A/G PLUS-Agarose (Santa Cruz,

Dallas, USA) and primary anti-v-SRC clone 327 (Millipore). After boiling samples, proteins

from cell lysates were resolved on an 8%, reducing SDS-PAGE gel, and transferred to an

Immobilon-P membrane for Western Blot detection. The blots were blocked with 1x Tris-buffer

saline (TBS), 5% skim-milk powder and 0.5% Nonidet P-40 (BioShop, Burlington, Canada).

Primary antibodies were diluted in 5% milk and 0.5% Nonidet P-40. Anti-GAPDH (Millipore),

anti-PTK2B (BD Transduction Laboratories, Mississauga, Canada) and anti-c-SRC GD11

(Millipore) were used to probe the blots. To measure phospho-c-SRC (Tyr419, BD Transduction

Laboratories) or phospho-PTK2B (Tyr402, New England Biolabs, Whitby, Canada), blots were

stripped and then blocked with 1x TBS, 5% w/v BSA and 0.1% Tween-20. After washing,

secondary goat anti-mouse HRP (Bio-Rad, Mississauga, Canada) or goat anti-rabbit HRP (Bio-

Rad) antibody was added, followed by enhanced chemiluminescent (ECL) detection (GE

122

Healthcare). PTK2B and c-SRC protein levels were determined by comparing their densitometry

to normalizer GAPDH, with the Molecular Imager GelDoc XR+ Imagin System (Bio-Rad).

siRNA Knockdown Experiments: Pools of four siRNA duplexes, specific for human c-SRC

(cat # M-003175-03) or PTK2B (cat # M-003164-02) mRNA, and a control pool of non-

targeting siRNA (cat # D-001206-13-05) were ordered from Dharmacon RNAi technologies

(siGENOME SMART Pool, Thermo Scientific, Lafayette, USA). With enriched CD4+ T-cells,

siRNA knockdown was performed using the Amaxa® Human T-cell Nucleofector

® Kit (Lonza,

Cologne, Germany), following the manufacturer’s instructions. Enriched T-cells (1.0 x 107)

were resuspended with 100 μL of Nucleofector solution, 900 nM of siRNA and 2 μL of siGuard

RNase inhibitor® (Genlantis, San Diego, USA), then electroporated with program V-024. Cells

were grown in 2 mL media lacking activators in a 37°C incubator for 12 hr, spun down, and

resuspended in 2 mL complete media containing activators. Forty-eight hours post-transfection,

cells were prepared for HIV-1 infection, harvested to measure protein knockdown of either c-

SRC or PTK2B by Western blot densitometry, or prepared for FACS staining.

qPCR Amplification of HIV-1 cDNA: Genomic and viral cDNA was isolated from 5 x 105

CD4+ T-cells, pre-treated by siRNA transfection and infected with HXB2 or JR-FL virus for 1 or

12 hours, using the QIAamp DNA Blood Mini Kit (Qiagen, Toronto, Canada). Samples were

heated at 56°C for 2 hours in QIAamp DNA Blood Mini Kit buffer AL to deactivate viable viral

particles.

The primer sets used to detect human or viral DNA (synthesized by the Center for Applied

Genomics, The Hospital for Sick Children, Toronto, Canada) were as follows: β-globin forward,

5′-CCCTTGGACCCAGAGGTTCT -3′; β-globin reverse, 5′-CGAGCACTTTCT TGCCATGA-

3′; early reverse transcripts (early RT) forward, 5′-GTAACTAGAGATCCCTCAG

ACCCTTTTAG-3′; early RT reverse, 5′-TAGCAGTGGCGCCCGA-3′; late reverse transcripts

(late RT) forward, 5′-CCGTCTGTTGTGTGACTCTGG-3′; late RT reverse, 5′-GAGTCCTGCG

TCGAGAGATCT-3′; 2-LTR circles forward, 5′-CCCTCAGACCCTTTTAGTCAGTG-3′; 2-

LTR circles reverse, 5′-TGGTGTGTAGTTCTGCCAATCA-3′; genomic Alu forward, 5′-GCCT

CCCAAAG TGCTGGGATTACAG-3′; HIV-1 gag reverse, 5′-GCTCTCGCACCCATCTCTCT

123

CC-3′; HIV-1 LTR forward, 5′-GCCTCA ATAAAGCTTGCCTTGA-3′; HIV-1 LTR reverse, 5′-

TCCACACT GACTAAAAGGGTCTGA-3’ [159, 542, 543]. For each qPCR run, 50-100 ng

template DNA (with the exception of the no-template control, NTC) was added to PCR tubes

containing: 12.5 μL 2x SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK),

300-900 nM of each forward and reverse primers, and PCR grade H2O (Roche Diagnostics,

Indianapolis, USA) up to a final volume of 25 μL. PCR cycling parameters were as follows:

initial denaturation at 95°C for 10 min; 40-50 cycles of amplification of 95°C for 15 sec, 58°C

for 30 sec (β-globin or late RT, 54°C for early RT or 2-LTR) and 60°C for 30 sec. To measure

integrated virus by Alu-gag PCR [542], tubes were first pre-amplified with 100 nM Alu forward

and 600 nM gag reverse primers, with 2x SYBR Green PCR Master Mix as above, for 10 cycles

of 93°C for 30 sec, 50°C for 60 sec, and 60°C for 100 sec. Nested PCR was performed on these

samples by adding 300 nM each of HIV-1 LTR forward and reverse primers, with 2x SYBR

Green PCR Master Mix, for 40 more cycles using the above parameters. Duplicate reactions

were analyzed using the Rotor-Gene RG-3000 thermocycler (Corbett Research, Montreal,

Canada). Five biological replicates in the c-SRC and PTK2B siRNA-treated groups were

compared to the non-targeting siRNA-treated group by the 2-ΔΔCT

comparative CT method [544].

Absolute quantification was performed for PCR amplicons of integrated virus, relative to the

chronically infected 8E5 cell line carrying one integrated proviral copy (NIH AIDS Reagent

Program, Dr. Thomas Folks).

In-Vitro Reverse Transcriptase Assay: Quantification of reverse transcriptase activity from

infected cell lysates was performed using the SYBR Green I qPCR-based product-enhanced RT

(PERT) assay [556]. Briefly, 5 x 105 CD4

+ T-cells were infected with HXB2 or JR-FL virus for

1 hr, washed twice with PBS, lysed, diluted 10-fold with 10x sample dilution buffer, and given 1

μg of MS2 RNA template (Roche Diagnostics) to reverse transcribe in vitro. The cDNA product

was measured by qPCR, and a standard curve of recombinant HIV-1 RT (Calbiochem) was used

to assign enzyme activity units to reverse transcriptase from the infected cell lysate samples.

Statistical Analysis: Means were compared using a two-tailed, unpaired Student’s t test and

corrected for multiple comparisons when more than two means were considered in an

experiment, to minimize the probability of type 1 errors. FACS data shows the percent positive

124

cells collected with CellQuest software (BD Biosciences) and analyzed by FlowJo version X

software. For all figures, an asterisk (*) denotes a p value < 0.05, two asterisks (**) denotes a p

value < 0.01 and three asterisks (***) denotes a p value < 0.001. Error bars shown are the

standard error around the mean (SEM).

125

3.4 Results

Kinase inhibitors targeting c-SRC significantly reduce HXB2 or JR-FL infection in

activated CD4+ T-lymphocytes

To confirm and extend the finding that dasatinib inhibits HIV-1 production [554], we

investigated whether pharmacological inhibitors of the SRC-family kinases, approved or under

clinical investigation for the treatment of various cancers, could inhibit early HIV-1 infection of

X4 or R5 luciferase-reporter viruses. For this purpose we tested dasatinib, saracatinib, a

SRC/ABL ATP reversible inhibitor in a phase II clinical trial for the treatment of advanced

ovarian cancer (http://clinicaltrials.gov/ct2/show/NCT00610714); KX2-391, a SRC substrate

competitive inhibitor in a phase II clinical trial evaluating efficacy in gastric and breast cancers

(http://clinicaltrials.gov/ct2/show/NCT01764087); and SRC Inhibitor-1, a dual ATP and

substrate competitive inhibitor in pre-clinical studies [557].

We first isolated peripheral blood mononuclear cells (PBMCs) from the whole blood of healthy

human donors, and could enrich a CD3+CD4

+ cell population to 98% purity with a negative

selection human CD4+ T-cell enrichment kit (Fig. 3.1a). No increase in apoptosis or necrosis

was observed after treating activated CD4+ T-cells with the four SRC inhibitors at the

concentrations tested (Fig. 3.2). Following drug-treatment and activation of primary CD4+ T-

cells for 24 hrs, we measured a strong decrease in luciferase activity after two days of HXB2

(X4) or JR-FL (R5) viral infection (Fig. 3.3). The dasatinib treatment exhibited the greatest

potency at doses in the 10-100 nM range.

126

Fig. 3.1: Cell Purity and Cell Viability of Enriched CD4+ T-Cells nucleofected with siRNA.

PBMCs were isolated from a healthy human donor by the Ficoll gradient method. CD4+ T-cells

were then enriched using the EasySep negative selection human CD4+ T-cell enrichment kit. (a)

One million cells were prepared for FACS and stained with Propidium Iodide (PI), CD4-APC

and CD3-PE. Cells were gated on the PI negative population. This FACS plot is representative

of three independent experiments. (b) Cell viability was assessed by 7-AAD staining 48 hrs

post-siRNA knockdown, on untreated CD4+ cells (un.), electroporated cells (mock), and cells

transfected with siRNA. Displayed is the average of two independent experiments, with the

error bars showing the range.

127

Fig. 3.2: SRC-Family Drug Inhibitors do not Increase Necrosis or Apoptosis of Activated

CD4+ T-Lymphocytes. Enriched CD4

+ T-cells isolated from a healthy donor were given one of

four SRC-family inhibitors (dasatinib, saracatinib, KX2-391, SRC Inhibitor-1) or DMSO solvent

(-ve control), and activated in growth media for 24 hrs. One million cells were then prepared for

FACS and stained with Propidium Iodide and Annexin V-FITC. No increase in

necrosis/apoptosis was observed for most drug treatments, relative to DMSO control. Ten μM of

KX2-391 or SRC Inhibitor-1 showed a slight increase in both PI and Annexin V stained cells,

suggesting increased late apoptotic cells.

128

Fig. 3.3: Pre-treating CD4+ T-cells with c-SRC kinase inhibitors prior to HXB2 or JR-FL

infection. Enriched CD4+ T-cells were administered DMSO solvent (-ve control) or one of four

SRC-family kinase inhibitors (dasatinib, saracatinib, KX2-391 or SRC Inhibitor-1), and grown in

growth media containing activators for 24 hrs. Cells (2 x 105) in 200 μL clear media were

infected with nef deficient, luciferase reporter viruses: 1.0 ng of HXB2 (a) or 26 ng of JR-FL (b).

After 48 hrs, cells were lysed and luciferase activity was detected. The means of all drug

treatments were compared with the mean of the DMSO solvent treatment. Five biological

replicates were run in each treatment. Error bars are standard error of the mean.

129

c-SRC and PTK2B become activated and co-immunoprecipitate shortly after HXB2 or JR-

FL infection

Given the SRC-family kinase inhibitor findings, we asked whether c-SRC signaling via PTK2B

plays an important role during early viral entry. c-SRC protein expression was strongly induced

in CD4+ T-cells 48 hrs after stimulating with anti-CD28, anti-CD3 and IL-2 (Fig. 3.4a). PTK2B

expression remained unaffected during T-cell activation. Meanwhile c-SRC became

phosphorylated at Tyr419 1 hr following HXB2 (X4) or JR-FL (R5) infection, a key

phosphorylation step that leads towards high c-SRC activity [558]. PTK2B also became

phosphorylated at Tyr402 after 1 hr of infection, suggesting SRC-family phosphorylation of

PTK2B [559]. Immunoprecipitation of c-SRC followed by probing for phosphorylated PTK2B

revealed the two kinases strongly co-associate after 1 hr of HXB2 or JR-FL infection, and that

PTK2B has been cleaved into a smaller fragment (Fig. 3.4b).

130

Fig. 3.4: c-SRC and PTK2B activation shortly after HIV-1 infection. (a), Western blot of

protein expression and phosphorylation state of c-SRC and PTK2B from the cell lysate of 1

million enriched CD4+ T-cells. Purified recombinant human pp60c-SRC protein was loaded in

lane 1. Cell lysates were isolated from PBMCs of a healthy donor (lane 2), activated for 48 hrs

(2 μg/mL CD28bHu, 10 μg/mL UCHT-1 and 10 units/mL recombinant IL-2, lane 3) and infected

with HXB2 or JR-FL virus for 1 hr (lanes 4 and 5). (b), Immunoprecipitation of c-SRC protein

from CD4+ T-cell lysate, followed by probing the Western blot with anti-Y402 PTK2B.

131

c-SRC and PTK2B siRNA knockdown reduce the infection of luciferase reporter viruses

and replication competent viruses

To test whether siRNA knockdown of either kinase reduces viral infection, we electroporated

siRNA into primary CD4+ T-cells, performed activation, and measured protein knockdown

48 hrs thereafter (Fig. 3.5a-b). We could consistently reduce c-SRC expression by 40% and

PTK2B expression by 80%. Cell viability at this time-point ranged from 70-80% (Fig. 3.1b). We

then infected the siRNA-transfected, activated CD4+ T-cells with HXB2 virus, and observed

decreased luciferase activity in c-SRC siRNA-treated, but not in PTK2B siRNA-treated cells,

relative to the non-targeting siRNA control (Fig. 3.5c). In contrast, siRNA knockdown of either

c-SRC or PTK2B reduced the luciferase activity following JR-FL viral infection (Fig. 3.5d).

This suggests that the co-receptor engaged upon entry (CXCR4 or CCR5) affects downstream

signaling events, with c-SRC common to the entry of both viruses tested and PTK2B playing a

role primarily during JR-FL (R5) infection. Further supporting this evidence, we also found that

knockdown of only c-SRC reduced viral integration of replication competent X4 IIIB, while

knockdown of either kinase reduced integration of R5 viruses Ba-L and clinical isolate

KNH1207 (Fig. 3.5e). Indeed, the unique response of X4 and R5 viruses to c-SRC or PTK2B

siRNA also affected the later stages of the replication cycle of IIIB or Ba-L, as determined by

p24 detected in cell-free supernatant three days post-infection (Fig. 3.5f-g).

132

Fig. 3.5: Luciferase reporter activity, viral integration and p24 production after c-SRC or

PTK2B siRNA knockdown. (a), c-SRC and PTK2B protein knockdown 48 hrs after

electroporating 1.0 x 107 enriched CD4

+ T-cells with 900 nM of non-targeting (NT), c-SRC- or

PTK2B-targeting siRNA. (b), Densitometry of protein knockdown relative to GAPDH from

panel A. (c and d), Forty-eight hours post-siRNA nucleofection and T-cell activation, 2 x 105

enriched CD4+ T-cells were infected with 1.0 ng of HXB2 or 26 ng of JR-FL. Luciferase activity

was measured from cell lysates two days thereafter. (e), In separate experiments, 2 x 105

enriched CD4+ T-cells were infected with replication competent X4 IIIB (1.1 ng), R5 Ba-L (43

ng) or R5 clinical isolate KNH1207 (16 ng). Absolute copy number of integrated provirus was

measured 12 hrs post-infection by qPCR. (f and g), p24 detection three days after IIIB or Ba-L

infection in cells nucleofected with siRNA. The means shown are of five biological replicates.

Error bars are standard error of the mean.

133

siRNA knockdown of c-SRC or PTK2B did not reduce surface expression of receptors

required for viral entry or reverse transcriptase activity

We then determined whether c-SRC or PTK2B siRNA knockdown altered the surface expression

of CD4 or the chemokine co-receptors, CXCR4 or CCR5, which are necessary for HIV-1

binding and fusion. Surface expression of CD4, CXCR4 and CCR5 was not affected by siRNA

knockdown of either kinase (Fig.3.6a-b). We then infected the siRNA-transfected, activated

CD4+ T-cells with HXB2 or JR-FL virus. After measuring reverse transcriptase (RT) activity

1 hr post-viral infection, we found no significant changes in RT activity following c-SRC or

PTK2B siRNA knockdown, relative to the non-targeting siRNA control (Fig. 3.6c-d).

134

Fig. 3.6: Cell surface receptor expression and RT activity. (a and b), T-cell surface expression

of CD4, CXCR4 and CCR5 48 hrs post-siRNA nucleofection, gating on 7-AAD- cells. (c and d),

5 x 105 siRNA-treated and activated CD4

+ T-cells were infected with 1.0 ng of HXB2 or 26 ng of

JR-FL in 200 μL of growth media containing activators. Cell lysates were collected after 1 hr to

measure in vitro reverse transcriptase activity. The means shown are of five biological replicates.

Error bars are standard error of the mean.

135

Suppressing c-SRC or PTK2B with siRNA reduced viral integration and increased late

reverse transcripts and 2-LTR circles

Next we explored at which stage following reverse transcription c-SRC and PTK2B are exerting

their effects, by measuring viral synthesis of early reverse transcripts (Early RT), late reverse

transcripts (Late RT), integrated virus in the host genome, and two-long terminal repeat (2-LTR)

circular DNA, which are indicative of failed integration [56]. Primer efficiency curves, time-

course experiments to optimally measure viral cDNA, and qPCR product melting curves are

presented in Figs. 3.7 and 3.8. We observed a 4-fold reduction in integration of either HXB2 or

JR-FL viruses in the c-SRC siRNA-treated cells (Fig. 3.9a-b). Surprisingly, this was associated

with increased early reverse transcripts, late reverse transcripts, as well as 2-LTR circles. These

experiments are the average of three separate donors (see Fig. 3.10 for the three independent

experiments). PTK2B siRNA knockdown also showed a similar trend as c-SRC siRNA

knockdown during JR-FL infection, but not with HXB2 infection. This is consistent with the

luciferase findings in Fig. 3.5c-d. The qPCR data suggest multiple interactions of both kinases

during HIV-1 entry, as we observed decreased integration that coincides with increased early

transcripts, late reverse transcripts and 2-LTR circles. The increase in late reverse transcripts

caused by c-SRC or PTK2B siRNA knockdown was not due to increased reverse transcription

(Fig. 3.6c-d), but rather, taken together with the observed increase in 2-LTR circles and decrease

in integrated provirus, that both kinases likely have a role in pre-integration complex (PIC)

formation or nuclear import of viral cDNA. Their nuclear affects on 2-LTR circles and

integration are akin to the integrase inhibitor raltegravir in HXB2 or Ba-L infection (Fig. 3.9c).

Interestingly, JR-FL virus appears resistant to high-dose raltegravir treatment, despite being

susceptible to c-SRC or PTK2B siRNA knockdown and SRC-family kinase inhibitors (Fig.

3.1b).

136

Fig. 3.7: qPCR and PERT Assay Standard Curves. To satisfy the assumptions of the 2-ΔΔCT

comparative CT method [544], primer efficiency curves were performed on serial dilutions of

DNA from CD4+ T-cells infected with HXB2 virus. Primer sets were tested for β-globin (a),

early reverse transcripts (b), late reverse transcripts (c), 2-LTR circles (d) and integrated virus

(e). A standard curve of DNA extracted from 8E5 cells was also constructed to convert cycle

threshold into copies of integrated virus (f). For the PERT assay, a standard curve of

recombinant reverse transcriptase (RT) was used to assign enzyme activity units to reverse

transcriptase in infected cell lysate samples (g). It was expected the efficiency curve of

recombinant RT would be less than 1.00, as the RNAse inhibitor added to prevent MS2 RNA

degradation in the PCR reaction is a known reverse transcriptase inhibitor [546]. Each data point

is the mean of technical triplicate PCR reactions. Red data points are DNA concentrations

beyond the linear range of qPCR detection.

137

Fig. 3.8: Time-Course and qPCR Melting Curves of HIV-1 DNA Targets. 5 x105 CD4

+ T-

cells were activated for 48 hrs and then infected with 1.0 ng of HXB2 or 26 ng of JR-FL in

complete media containing activators. A-D, Early reverse transcripts (Early RT), late reverse

transcripts (Late RT), 2-LTR circles and integrated HIV-1 were measured in DNA extracted

from cell lysates 0.5-48 hrs post-infection. Time points in grey represent when each transcript

was measured in subsequent experiments. E-H, Representative melting curves of qPCR

products (blue) relative to no template control reactions (NTC, black) demonstrate the specificity

of the published primer sets. The heterogeneous mixture of Alu-gag amplified sequences during

the exponential phase explains the various melting peaks seen after the kinetic phase of nested

PCR in panel H.

138

Fig. 3.9: qPCR of early HXB2 or JR-FL infection after c-SRC or PTK2B siRNA

knockdown. (a and b), Forty-eight hours post-siRNA nucleofection and T-cell activation, 5 x 105

enriched CD4+ T-cells were infected with 1.0 ng of HXB2 or 26 ng of JR-FL. Cells were lysed

after 12 hrs of infection to measure early reverse transcripts (Early RT), late reverse transcripts

(Late RT), 2-LTR circle formation or integrated virus. qPCR of HXB2 or JR-FL DNA was

normalized to genomic β-globin, and graphed relative to the non-targeting (NT) siRNA treatment

(x-axis). The means shown are the average of three different donors from three independent

experiments (Fig. 3.10 shows these separately). (c), Enriched CD4+ T-cells (2 x 10

5) were

incubated with 20 μM raltegrevir 24 hours prior to infection with HXB2, Ba-L or JR-FL. qPCR

products of HIV-1 were measured as in panels a and b. Error bars are standard error of the mean.

139

Fig. 3.10: qPCR of HXB2 or JR-FL Infection in CD4+ T-Cells from Three Separate

Donors. Three independent experiments of HXB2 or JR-FL infection measured by qPCR.

CD4+ T-cells from three donors were isolated, and were electroporated with 900 nM of non-

targeting (NT), c-SRC or PTK2B siRNA. Cells were activated in complete media 12 hrs later,

and at 48 hrs post-transfection, 5 x 105 T-cells were infected with 1.0 ng of HXB2 (a-c) or 26 ng

of JR-FL (d-f). Early reverse transcripts (E. RT) and late reverse transcripts (L. RT) were

measured after 1 hr of infection, while 2-LTR circles (2-LTR) and integration (Int. HIV-1) were

quantified from cell lysates collected after 12 hrs of infection. All qPCR targets were normalized

to genomic β-globin, and cells treated with NT siRNA were the reference treatment set to 1-fold

(x-axis). The means of c-SRC or PTK2B siRNA treatments were compared with the NT siRNA

treatment, for a given HIV-1 qPCR target. Technical duplicates were performed, and means are

the average of five biological replicates. Error bars are standard error of the mean.

140

3.5 Discussion

Efficacious and well-tolerated therapeutics that reduce virological and immunological failure are

key to designing new HIV-1 regimens. Our study demonstrates a new means to inhibit HIV-1

integration and modulate early HIV-1 entry in CD4+ T-lymphocytes by targeting specific host

non-receptor tyrosine kinases (summary schematic Fig. 3.11). Our work herein is the first report

to show a specific role for the protein tyrosine kinase, c-SRC, in facilitating HIV-1 infection of

X4 and R5 viruses.

Fig. 3.11: Summary schematic of the proposed mechanism for c-SRC involvement during

early HIV-1 infection.

141

We first demonstrate that four SRC-family kinase inhibitors reduced HXB2 or JR-FL infection

by as much as 90% in activated CD4+ T-cells (Fig. 3.3). The diversity of mechanisms by which

these four drugs inhibit c-SRC, and differential on- and off-target efficacy, provide a strong

rationale for testing these inhibitors for their ability to reduce in vivo HIV-1 infection in a

humanized mouse model. While preclinical-stage inhibitors targeting other host kinases have

been found to influence viral infection (c-Jun N-terminal Kinase (JNK) inhibitors that reduce

integrase stability, protein kinase C agonists that reactivate latent HIV-1 provirus to purge latent

reservoirs [560]), our study is the first to describe mechanisms by which clinically approved (in

the case of dasatinib), safe, and well-tolerated SRC-family inhibitors act on early HIV-1

infection, suggesting these inhibitors could be promptly repurposed for potential treatment of

HIV-1 infection. For instance, a 75 nM dose of dasatinib has recently been shown to strongly

inhibit p24 production in activated CD4+ T-cells cultured from HIV

+, treatment-naïve patients

[554]. This dasatinib concentration was below the well-tolerated in vivo level of 180 nM [561],

and Pogliaghi et al. reported no increase in cell toxicity in cultured CD4+ T-cells [554].

Moreover, dasatinib has recently been shown to synergistically enhance the antiviral properties

of sofosbuvir, further inhibiting in vitro hepatitis C infection [532]. It also has potency in

inhibiting dengue virus 2 RNA replication by inhibiting the SRC-family kinase member FYN

[178].

To investigate potential targets of these four SRC kinase inhibitors, we demonstrated c-SRC and

its binding partner PTK2B become activated within 1 hr of HXB2 or JR-FL infection, and that

phosphorylated PTK2B co-immunoprecipitates with c-SRC after infection (Fig. 3.4). Activity of

calpains, which are Ca2+-dependent proteases known to cleave PTK2B at focal adhesions [336],

explains why a smaller PTK2B fragment immunoprecipitated with c-SRC in Fig. 3.4B. The

activation or autophosphorylation of cleaved PTK2B at tyrosine 402 may be induced in response

to chemokine engagement [147] or activation of the purinergic receptor P2Y2 during early HIV-

1 infection [345]. This may form a signaling complex that contains c-SRC and actin interacting

proteins that facilitate early HIV-1 entry and subsequent formation of the reverse transcriptase

complex in the cytosol. We then showed that reducing c-SRC protein with targeted-siRNA

reduced the luciferase activity of HXB2 and JR-FL viruses, and decreased the copy number of

142

integrated IIIB, Ba-L or KNH1207 provirus (Fig. 3.5). c-SRC siRNA knockdown affects HIV-1

infection at a stage following reverse transcription and prior to genomic integration (Fig. 3.6 and

3.9). We also show a similar finding with PTK2B siRNA knockdown, reducing JR-FL luciferase

reporter activity and viral integration (Fig. 3.5d and 3.9b). However, PTK2B siRNA knockdown

had little effect on the entry of the X4 viruses HXB2 or IIIB (Fig. 3.5c, 3.5e and 3.9a). While

PTK2B activation has been reported to be downstream of both CXCR4 and CCR5 co-receptor

engagement with gp120/gp41 in a T-cell line [147], our finding suggests that PTK2B signaling is

more dispensable during HXB2 (X4) infection in primary CD4+ T-cells. It is possible that

engagement of CXCR4, abundantly expressed on activated CD4+ T-cells compared to CCR5

(Fig. 3.6a-b), may provide a more robust signal that causes increased activation of c-SRC

independently of PTK2B activation.

It is intriguing that reducing either kinase had no effect on JR-FL reverse transcription (Fig.

3.6d) yet inhibited viral integration (Fig. 3.9b). Meanwhile, the same siRNA knockdown

conditions increased reverse transcription and viral integration in our previous report on

transformed T-cell lines [555]. Jurkat T-cells show constitutive c-SRC and PTK2B expression

and activation, as well as other signaling abnormalities downstream of the T-cell receptor [562],

suggesting a very different intracellular environment compared with activated primary CD4+ T-

cells. Indeed, our work is adding to a growing body of evidence that the involvement of receptor

and non-receptor tyrosine kinases during HIV-1 infection is highly dependent on the cell model

investigated [289, 563, 564], and that transformed cell lines may not be physiologically relevant

models of signal transduction during in vivo HIV-1 infection.

Our findings also suggest c-SRC signaling is a common feature of the early HIV-1 life cycle, and

that its interaction with viral and host proteins during PIC formation, viral cDNA nuclear

transport and genomic integration warrants further research. In light of the 20-fold increase in

late reverse transcripts following c-SRC siRNA knockdown (Fig. 3.9a), our findings offer the

potential to advance the study of host proteins integral to the PIC. Cellular kinases have been

reported to directly phosphorylate viral PIC proteins (reviewed in [155]) and assist with

integration into the host genome [154, 159]. Moreover, the identities of tyrosine kinases that

phosphorylate known viral constituents of the PIC remain unknown. For instance, the tyrosine

143

kinases that phosphorylate matrix protein p17 at C-terminal Y132 and preferentially target

matrix protein to the nucleus, have yet to be identified [158]. Future experiments using

knockdown of c-SRC or PTK2B could reveal whether these tyrosine kinases directly

phosphorylate host or viral PIC proteins, and could also uncover novel protein interactions for

pharmacological intervention. Whether nuclear c-SRC directly participates in HIV-1 integration

warrants further research. Moreover, c-SRC siRNA knockdown produced a viral qPCR signal

reminiscent of integrase inhibitors (Fig. 3.9c): reduced viral integration and increased 2-LTR

circles [565-567]. siRNA targeting PTK2B has also been shown to inhibit mutated HIV-1

strains that are resistant to reverse transcriptase or integrase inhibitors [345]. The unexpected

finding that JR-FL was resistant to raltegravir treatment opens up the possibility of c-SRC or

PTK2B inhibitors substituting for integrase inhibitors when combination therapies fail due to

integrase mutations. This warrants further experiments in humanized animal models to explore

how these drugs can improve existing HIV-1 treatment regimens.

In conclusion, our findings demonstrate the importance of host non-receptor tyrosine kinases in

the early stages of HIV-1 entry, specifically c-SRC and to a lesser extent PTK2B. Furthermore,

we demonstrate that these kinases can have multifunctional roles when interacting with the virus

at various stages of viral entry. The mechanism by which reducing or inhibiting c-SRC or

PTK2B restricts viral integration remains to be explored. Nonetheless, the availability of well-

studied SRC-family kinase inhibitors of known efficacy, specificity, pharmacokinetics and

toxicity profile, opens up new avenues for repurposing these drugs in combination with known

antivirals as novel means to inhibit HIV-1 and other viral infections. In particular, these may

prove useful for patients with contraindications for current HIV-1 drug combinations.

144

Chapter 4: A Rapid Screening Assay Identifies

Monotherapy with Interferon-ß and Combination Therapies

with Nucleoside Analogues as Effective Inhibitors of Ebola

Virus

Role of author McCarthy S.D.: Illustrated Figure 4.1, and performed all experiments for Figures

4.2, 4.3 and Figures 4.5-4.8. I measured % inhibition for the majority of Figure 4.4. I also

performed all experiments for Tables 4.1 and 4.2. I analyzed all the data, co-wrote the

manuscript draft, and performed experiments to address reviewer’s comments.

A version of this chapter was published in the Journal PLoS Neglected Tropical Diseases:

McCarthy, S.D., Majchrzak-Kita, B., Racine, T., Kozlowski, H.N., Baker, D.P., Hoenen, T.,

Kobinger, G.P., Fish, E.N., and Branch, D.R. 2016. A Rapid Screening Assay Identifies

Monotherapy with Interferon-ß and Combination Therapies with Nucleoside Analogues as

Effective Inhibitors of Ebola Virus. PLoS NTD, 10(1):e0004364.

145

4.1 Abstract

To date there are no approved antiviral drugs for the treatment of Ebola virus disease (EVD).

While a number of candidate drugs have shown limited efficacy in vitro and/or in non-human

primate studies, differences in experimental methodologies make it difficult to compare their

therapeutic effectiveness. Using an in vitro model of Ebola Zaire replication with transcription-

competent virus like particles (trVLPs), requiring only level 2 biosafety containment, we

compared the activities of the type I interferons (IFNs) IFN-α and IFN-ß, a panel of viral

polymerase inhibitors (lamivudine (3TC), zidovudine (AZT) tenofovir (TFV), favipiravir (FPV),

the active metabolite of brincidofovir, cidofovir (CDV)), and the estrogen receptor modulator,

toremifene (TOR), in inhibiting viral replication in dose-response and time course studies. We

also tested 28 two- and 56 three-drug combinations against Ebola replication. IFN-α and IFN-ß

inhibited viral replication 24 hours post-infection (IC50 0.038 µM and 0.016 µM, respectively).

3TC, AZT and TFV inhibited Ebola replication when used alone (50-62%) or in combination

(87%). They exhibited lower IC50 (0.98-6.2 μM) compared with FPV (36.8 μM), when

administered 24 hours post-infection. Unexpectedly, CDV had a narrow therapeutic window

(6.25-25 μM). When dosed > 50 µM, CDV treatment enhanced viral infection. IFN-ß exhibited

strong synergy with 3TC (97.3% inhibition) or in triple combination with 3TC and AZT (95.8%

inhibition). This study demonstrates that IFNs and viral polymerase inhibitors may have utility in

EVD. We identified several 2 and 3 drug combinations with strong anti-Ebola activity,

confirmed in studies using fully infectious ZEBOV, providing a rationale for testing combination

therapies in animal models of lethal Ebola challenge. These studies open up new possibilities for

novel therapeutic options, in particular combination therapies, which could prevent and treat

Ebola infection and potentially reduce drug resistance.

146

4.2 Introduction

As of December 13, 2015, the current outbreak of Ebola virus disease (EVD) in West Africa

resulted in 28,633 cumulative cases and 11,314 deaths [568]. Two potential vaccine candidates,

rVSV∆G-ZEBOV and ChAd3-EBO Z, have shown durable protection from lethal Ebola

challenge in mice [569] and macaques [32] respectively, and are part of the phase II/III

PREVAIL trial in Liberia and Guinea (https://clinicaltrials.gov/ct2/show/NCT02344407). Other

potential therapeutics, such as convalescent plasma and the antibody cocktail ZMapp [30] have

been approved for an emergency phase II/III trial in Guinea

(https://clinicaltrials.gov/ct2/show/NCT02342171) and a phase I trial in Liberia

(https://clinicaltrials.gov/ct2/show/NCT02363322), respectively. However, to date there is no

licensed vaccine or treatment for EVD, although improvements in supportive care are increasing

survival rates [570].

Repurposing antivirals used for other viral infections, based on knowledge of mechanisms of

action, has prompted accumulating interest in the application of different nucleoside/nucleotide

analogues and type I interferons (IFNs) for the treatment of Ebola virus disease (EVD).

Experimental nucleoside analogues may have therapeutic efficacy for EVD, given the evidence

of protection in primate and rodent disease models, 2-6 days after lethal Ebola or the related

hemorrhagic Marburg virus challenges [23, 418]. Favipiravir (FPV), a viral polymerase inhibitor,

provides 100% protection when administered 6 days after challenge with a lethal dose of Ebola

virus [23] and has been evaluated in the phase II/III JIKI trial in Guinea

(https://clinicaltrials.gov/ct2/show/NCT02329054). TKM-Ebola, a cocktail of siRNAs targeting

VP35 and L polymerase and brincidofovir (BCV), a viral polymerase inhibitor that has activity

against dsDNA viruses such as adenovirus and cytomegalovirus [571], were also considered for

treatment against EVD. The brincidofovir trial was halted, ostensibly because of projections of

low recruitment.

Despite infecting different target cells, Ebola and HIV-1 share many similar features early in

their replication cycle. Both are RNA viruses that package a viral polymerase (L for Ebola, RT

for HIV-1) required for early replication in the cytosol of the host cell [572]. Homology-based

147

structural prediction of the RNA-dependent RNA polymerase of Ebola indicates the polymerase

contains conserved structural motifs in the catalytic palm subdomain similar to viral DNA

polymerases [455], supportive of nucleoside analogues potentially inhibiting Ebola replication.

Inhibiting HIV-1 reverse transcription with nucleoside analogues such as lamivudine (3TC,

cytidine analogue), zidovudine (AZT, thymidine analogue) or tenofovir (TFV, adenosine

monophosphate analogue) is the basis for highly active antiretroviral treatment (HAART) [523,

573]. Nucleoside analogues are on the WHO list of essential medicines and can be deployed in

limited resource settings [574]. Moreover, AZT binds RNA through G-C and A-U bases [575],

prompting us to evaluate whether these nucleoside analogues might also inhibit Ebola

replication.

Type I IFNs mediate diverse biological effects, including cell type-independent antiviral

responses and cell type-restricted responses of immunological relevance. IFNs inhibit viral

infection by preventing viral entry into target cells and by blocking different stages of the viral

replication cycle for different viruses. Moreover, type I IFNs have a critical role in linking the

innate and adaptive immune responses to viral challenge. IFN-α/β expression occurs as the

earliest non-specific response to viral infection. Indeed, viruses have evolved immune evasion

strategies specifically targeted against an IFN response, confirming the importance of IFNs as

antivirals. This immune evasion strategy is relevant when one considers the IFN response to

Ebola infection [576]. Ebola proteins VP24 and VP35 inhibit host cell systems that lead to IFN

production and also inhibit events associated with an IFN response [577-579]. VP24 blocks the

binding of importins to phosphorylated STAT1, preventing STAT1 nuclear translocation

required for transcription of interferon simulated genes [577]. VP35 binds viral dsRNA,

preventing dsRNA degradation [578] and inhibits the phosphorylation of IRF-3 and the

SUMOylation of IRF-3 and IRF-7, thereby limiting IFN production [579]. Despite these virally-

encoded mechanisms to limit an IFN response to infection, different rodent and non-human

primate studies provide evidence for IFN-induced partial protection: the effects of IFN-α/β

treatment in lethal Ebola virus infection reduced viremia and prolonged survival [31, 580, 581].

Thus, a potential therapeutic effect for IFNs as monotherapy in EVD, or in combination with

other anti-Ebola therapies, has not been resolved.

148

4.3 Materials and Methods

Cell culture and trVLP infection: We employed an established mini-genome system to rapidly

evaluate candidate drugs that could inhibit Ebola Zaire replication under BSL2 conditions (see

Fig. 4.1 for an an illustration of the reporter system) [534]. The mini-genome encodes 3 of the 7

ZEBOV proteins (VP24, VP40 and GP1,2) and a luciferase reporter gene. Expression plasmids

for the remaining four Ebola nucleocapsid proteins (L, NP, VP30 and VP35) were also included

during transfection. Cell culture conditions and virus infections were performed as previously

described [534]. Briefly, 80,000 producer 293T cells (American Type Culture Collection;

ATCC, Rockville, USA) were seeded in individual wells of 24-well plates in 400 μL Dulbecco’s

Modified Eagle Medium (DMEM) containing 10% FBS, 1% penicillin and 1% streptomycin,

and grown in 5% C02 atmosphere at 37°C. Cells were transfected with the viral replication

protein plasmids (L, NP, VP30, VP35), a tetracistronic Ebola mini-genome and the T7

polymerase, using the CalPhos Mammalian Transfection Kit (Clontech Laboratories). Twenty-

four hours later, medium was replaced with 800 μL DMEM with 5% FBS. The replication and

transcription-competent virus like particles (trVLPs) were harvested 3 days later. Virus stock

was frozen at -80°C.

For infection, 293T target cells were seeded at 80,000 cells in 400 μL of DMEM supplemented

with 10% FBS. Target cells were then transfected with the four viral replication protein

plasmids, as well as Tim-1, to allow efficient virus binding and entry. Twenty-four hours post-

transfection, 25 μL of trVLP stock was diluted in 600 μL of DMEM with 5% FBS, warmed to

37°C for 30 min, then added to target cells. Medium was removed the following day and

replaced with 800 μL DMEM with 5% FBS. Four days post-infection, the medium was

aspirated and cells were re-suspended in 200 μL of 1x Renila Luciferase Assay Lysis Buffer

(Renilla Luciferase Assay System, Promega). Lysates were assayed for luciferase activity.

ZEBOV-eGFP infection: We generated recombinant ZEBOV expressing enhanced green

fluorescent protein (eGFP) from cDNA clones of full-length infectious ZEBOV, as previously

described [582]. The eGFP reporter protein was expressed as an eighth gene, and the virus

exhibited an in vitro phenotype similar to wild type ZEBOV. Notably, in vivo, incorporation of

149

Fig. 4.1: Transcription and replication competent virus-like particle (trVLP) assay.

ZEBOV trVLP particles were collected from the supernatant of 293T producer cells. These

virus particles carried tetracistronic RNA genomes that contain a renila luciferase reporter gene,

VP40, GP1,2 and VP24. Target 293T cells were transcfected with expression plasmids prior to

infection: Tim-1 to allow efficient viral entry and RdRP L, NP, VP30 and VP35 to facilitate viral

replication. Cytosolic expression of these four additional viral proteins allowed formation of the

RNA-dependent RNA replication complex after receptor-mediated endocytosis of trVLP virus.

Luciferase activity detected in cell lysates indicated successful viral transcription and translation

of the ZEBOV mini-genome.

150

GFP into wild type ZEBOV results in some attenuation of disease [582]. All work with

infectious ZEBOV was performed in biosafety level 4 (BSL4), at the National Microbiology

Laboratory of the Public Health Agency of Canada in Winnipeg, Manitoba. 293T cells (30,000)

were seeded in 96-well plates in 100 μL DMEM with 10% FBS. Twenty-four hours thereafter,

the medium was replaced with 100 μL DMEM with 10% FBS containing ZEBOV-GFP at an

MOI of 0.1. Twenty-four hours post-infection, the medium was removed and replaced with 200

µL of DMEM with 5% FBS, or 190 μL DMEM with 5% FBS and 10 μL of single or

combinations of drugs. eGFP fluorescence was measured 3 days post-infection using a Synergy

HTX Multi-Mode Microplate Reader (BioTek).

Drugs: For these experiments, we used toremifiene citrate (TOR; Sigma), cidofovir hydrate

(CDV; Sigma) favipiravir (FPV, T-705; Cellagen Technology), lamivudine (3TC; Sigma)

zidovudine (AZT), tenofovir (TFV) maraviroc (MVC; NIH AIDS Reagent Program), Infergen

(IFN alfacon-1, Pharmunion Bsv Development Ltd.) or human interferon beta-1a (IFN-β,

Avonex; Biogen).

Mini-genome RNA extraction and qRT-PCR quantification of viral RNA: Forty-eight hours

after trVLP infection, medium was aspirated from 293T cells that had either been left untreated

or treated with the various drugs and total RNA extracted from cell lysates with 500 μL of

TRIzol (Thermo Fisher Scientific). cDNA synthesis was performed on 5 µg of total RNA, using

the First-Strand cDNA Synthesis Kit (GE Healthcare Life Sciences), according to the

manufacturer’s instructions. A 20 µl reaction also contained bulk first-strand cDNA reaction

mix, DTT solution and 40 pmol of one of two trVLP specific primers [583]: vRNA forward (5’-

GGC CTC TTC TTA TTT ATG GCG A -3’), or cRNA/mRNA reverse (5’-AGA ACC ATT

ACC AGA TTT GCC TGA-3’). Both primers were synthesized by the Center for Applied

Genomics (The Hospital for Sick Children, Toronto, Canada). Real-time qPCR reactions (25 µl)

were conducted in duplicate, using the Rotor-Gene RG-3000 thermocycler (Corbett Research,

Montreal, Canada). Each reaction contained 100 ng template cDNA, 12.5 µL 2 x SYBR Green

PCR Master Mix (Applied Biosystems, Warrington, UK), 300 nM of both the forward (vRNA)

and reverse (cRNA/mRNA) primers, and PCR grade H2O (Roche Diagnostics, Indianapolis,

USA). Samples lacking reverse transcriptase (No RT) during first-strand cDNA synthesis served

151

as negative controls. Cycling parameters were as follows: initial denaturation at 95°C for 10

min, followed by 40 cycles of amplification with 95°C for 15 seconds, 56°C for 30 seconds, and

60°C for 30 seconds. Biological triplicates in the drug-treated groups were normalized to the

average Ct of infected cells given DMSO solvent alone, by the 2-ΔCT

comparative CT method.

Cell viability assay: Dose-response cytotoxicity/viability assays were conducted in 293T cells 4

days post-infection for each of the drugs examined, either alone or in the various combinations

indicated, using the MTT assay as previously described [584].

Statistics: Means were compared using a two-tailed, unpaired Student’s t test and corrected for

multiple comparisons. For all figures, (*) denotes a p value <0.05, (**) denotes a p value <0.01

and (***) denotes a p value <0.001. Error bars shown are the standard error around the mean

(SEM). Synergy between two and three-drug combinations and combination index (CI) were

calculated with CompuSyn Version 1.0 [533]. The coefficient of determination (R2) was

determined for simple linear regressions.

152

4.4 Results

We employed an established mini-genome system to rapidly evaluate candidate drugs that could

inhibit Ebola Zaire replication under BSL 2 conditions [534, 535, 585]. At the outset, we

established the experimental conditions for infection with replication and transcription-

competent virus like particles (trVLPs), by examining luciferase activity under various

transfection and drug treatment conditions, which included transfection with viral support protein

plasmids (Fig. 4.2). We included treatment with maraviroc, a CCR5 inhibitor, that would have

no effect on trVLP entry and infection, thereby serving as a negative control for subsequent

treatment regimens. The triple combination of 3TC + AZT + TFV significantly inhibited trVLP

infection, and further inhibited luciferase activity of a second passage of 293T cells when

administered supernatant of a previous passage of cells.

In a second series of experiments, we examined the inhibitory effects of IFN-α (0.5 µM/10,000

U/mL), IFN-ß (0.2 µM/1,000 U/ml), TOR (5 µM), CDV (100 µM), FPV (100 µM), and a

combination of 3TC, AZT and TFV (5 µM each) on trVLP infection of 293T cells (Fig. 4.3).

Specifically, the 293T cells were treated with the different drugs at four different times relative

to infection with trVLP, as indicated. We provide evidence that for each of the individual drugs

and for the triple drug combination, at the doses indicated, trVLP infection of 293T cells is

inhibited when treatment is initiated at +2, +6 or +24 hours post-infection. Interestingly, TOR,

an estrogen receptor modulator discovered in a high throughput screen as a potent inhibitor of

Ebola [25], significantly reduced viral luciferase activity at all time-points tested. For IFN-α,

IFN-ß, TOR and FPV treatments, maximal inhibition of trVLP infection was achieved when the

cells were treated prior to challenge with trVLP. By contrast, pre-treatment with CDV at 100

µM, 24 hours prior to infection with trVLP, resulted in enhanced infection.

153

Fig. 4.2: IFNs and nucleoside analogues inhibit Ebola mini-genome replication in vitro. (A)

293T cells were either left untreated (-), transfected with Tim-1 and the viral support protein

plasmids NP, VP30, VP35, but not L (-L), or transfected with Tim-1 and all support plasmids

that permit Ebola mini-genome entry and replication (+). At -24, 0, and +24 hours relative to

infection, cells were treated with 5 μM of maraviroc (MVC), lamivudine (3TC), zidovudine

(AZT), or tenofovir (TFV), or combinations of the three nucleoside analogues. Luciferase

activity was measured four days after infection with trVLPs. (B) Luciferase activity of trVLP

producer cells (0), first passage of 293T cells (1) treated with nucleoside analogues, and second

passage of 293T cells (2) treated with nucleoside analogues. (C) Luciferase activity of 293T cells

after treating with 5 μM combination of nucleoside analogues, cidofovir (CDV) or favipiravir

(FPV). (D) Luciferase activity of 293T cells treated with IFN-α or IFN-β. The values are means

of four biological replicates and are representative of two independent experiments. Error bars

are the standard error of the mean. All drug treatment outcomes were statistically compared with

the (+) control group in panels A, C and D.

154

Fig. 4.3: IFNs, toremifene, and nucleoside analogues inhibit trVLP replication. 293T cells

were either left untreated (-), transfected with Tim-1 and the nucleocapsid plasmids NP, VP30,

VP35 but not L (-L), or transfected withTim-1 and all expression plasmids to permit Ebola mini-

genome entry and replication (+). (A-F) Cells were treated with the indicated drugs at the

indicated doses, at each of the time points shown. Luciferase activity was measured 4 days post-

trVLP infection. Values shown are the means of 4 biological replicates and are representative of

2 independent experiments. Error bars are the standard error of the mean. All drug treatment

outcomes were statistically compared with the (+) control group.

155

In subsequent dose-response studies, we compared the inhibitory effects of IFN-α, IFN-ß, TOR,

CDV, FPV, 3TC, AZT or TFV when administered 24 hours post trVLP infection (Fig. 4.4). The

data in Figure 4.4I summarize the IC50 dose for each drug. The IFNs exhibited the lowest IC50

values at 0.016 µM for IFN-ß and 0.038 µM for IFN-α. The data show a log-fold difference in

IC50 values for IFN-α and IFN-ß when compared in terms of U/ml, the norm for antiviral activity

measurements (Figure 4.4 A, B). TOR had the next lowest IC50 (0.36 µM) and completely

inhibited infection at doses > 5 µM (Figure 4.4C). TFV had an IC50 at 0.98 µM. CDV, 3TC and

AZT all exhibited similar IC50 values in the dose range 4.2-7.8 µM, while FPV had the highest

IC50 of the nucleoside analogues at 36.8 µM. At their IC50 concentration, none of these drugs

directly inhibited luciferase reporter activity (Fig. 4.5). We observed a relatively small antiviral

dose range for CDV (1.5-25 µM) (Figure 4.4D), beyond which the drug appeared to enhance

viral infection (Fig. 4.6). In cell viability assays we observe that at doses >10 µM CDV affects

cell viability, confounding the interpretation of the effects of CDV on viral replication.

156

Fig. 4.4: IFNs, toremifene and nucleoside analogues administered 24 hrs post-exposure

inhibit Ebola-mini-genome replication. 293T cells were either left untreated (-), transfected

with Tim-1 and the nucleocapsid plasmids NP, VP30, VP35 but not L (-L), or transfected with

Tim-1 and all expression plasmids to permit Ebola mini-genome entry and replication (+), as

described in Materials and Methods. Twenty-four hours post-trVLP infection, cells were either

left untreated, or treated with the indicated drugs (A-I) Dose-response plots for each of the

indicated drugs. Luciferase activity (black circles) or cell viability (white squares) was measured

4 days post-infection (3 days after drug treatment). Values are the means of 4 biological

replicates and are representative of 2 independent experiments. Error bars are the standard error

of the mean.

157

Fig. 4.5: No direct effect of drugs on Renilla luciferase reporter assay. 293T cells transfected

and then infected with trVLP were lysed 4 days post-infection. Cell lysate suspended in CCLR

reagent was aliquoted into separate tubes and spiked with each drug at the IC50 dose from Fig.

4.4I or DMSO solvent (+). Luciferase activity was then quantified. Values are the means of 3

independent experiments. Error bars are the standard error of the mean.

158

Fig. 4.6: CDV treatment increases trVLP replication. 293T cells were transfected with all

expression plasmids that permit Ebola mini-genome entry and replication, then treated with the

indicated doses of CDV 24 hrs post-trVLP infection. Luciferase activity was measured four days

after cells were infected (3 days post-CDV treatment). Values are the means of 4 biological

replicates and are representative of 2 independent experiments. Error bars are the standard error

of the mean. CDV treatment outcomes were compared with the (+) control group.

159

In an orthogonal assay to confirm these findings, we next measured viral replication and

transcription by qRT-PCR, following trVLP infection. trVLP-infected cells were either left

untreated, or treated with the different drugs 24 hours post-infection, then viral replication and

transcription evaluated 24 hours later (Fig. 4.7). All treatments, with the exception of TOR,

significantly reduced the amount of genomic vRNA detected within cells (Figure 4.7A) and all

treatments significantly reduced the synthesis of cRNA and mRNA isolated from infected cells

(Figure 4.7B). Notably, IFN-ß treatment of trVLP-infected cells resulted in the greatest

reduction in viral replication and transcription.

160

Fig. 4.7: IFNs, toremifene and nucleoside analogues reduce trVLP replication and

transcription. 293T cells were transfected with support plasmids and infected with trVLP.

Twenty-four hours post-infection, cells were treated with the indicated drugs at their IC50 doses,

determined from Figure 2I. At 48 hrs post-infection, total RNA was extracted, reverse

transcribed, then quantified by qPCR. Relative fold-change in negative sense vRNA transcripts

(A) and positive sense cRNA and mRNA (B) was compared with infected, untreated cells

(solvent, + control). Technical duplicates were examined by qPCR, and means are the average

of three biological replicates. Error bars are the standard error around the mean.

161

Next we examined the effectiveness of two and three drug combinations on trVLP infection. We

first examined 28 two-drug combinations, using each drug at its luciferase IC50 value, and used

the median-effect equation and combination index theorem [533] to determine drug synergy,

additive or sub-additive effects (Figure 4.8A). Synergy is defined as greater than additive effect

when drugs were combined (CI<1), additive as the effect expected when combining each drug

(CI=1) and sub-additive as a smaller than expected additive effect (CI>1). Fractional inhibition

(Fi) is defined as the percent reduction in luciferase activity. When administered 24 hours post-

infection, many of the two-drug combinations showed strong synergism in inhibiting trVLP

replication (Figure 4.8J), with IFN-β + 3TC demonstrating the greatest synergism (97.3%

inhibition, CI = 0.028). 3TC was synergistic with all seven other drugs tested. Notably, when

CDV was used in combination with FPV, AZT, TFV or IFN-α, it produced a sub-additive effect.

Next we tested all possible 56 three-drug combinations, using each drug at its IC50 value, to

assess whether adding a third drug enhanced efficacy compared with two-drug combinations

(Figure 4.8B-I) . This series of experiments served to validate our two-drug findings, as

synergistic two-drug combinations such as IFN-β + 3TC and IFN-β + AZT, predicted strong

synergy for the triple drug combination of IFN-ß + 3TC + AZT. As anticipated from the two-

drug polygonogram, CDV was sub-additive when combined in three-drug combinations (Figure

4.8E). This was most evident even when CDV was administered in conjunction with two-drug

combinations that had shown strong synergy, such as IFN-β + 3TC or FPV + TFV, further

indicating that CDV diminishes the antiviral effects of other drugs. IFN-ß, 3TC, AZT and TFV

all promoted strong synergism when included in triple drug combinations, with IFN-β + AZT

specifically providing strong synergism when combined in three unique triple therapies.

From these two-drug and three-drug screens, we calculated the combination index (CI) and

fractional inhibition (Fi) (Figure 4.8J-K). Many of the synergistic drug combinations (i.e. low

CI) included one nucleoside analogue and an IFN, while those drug combinations that were sub-

additive all included CDV. IFN-β was predominant in the most efficacious two- and three-drug

combinations. In particular, IFN-β + 3TC and IFN-β + 3TC + AZT consistently exhibited the

strongest synergism and highest Fi when administered 24 hours post-infection. Refer also to

Tables 4.1 and 4.2.

162

163

Fig. 4.8: 2 and 3 drug combinations synergistically inhibit Ebola trVLP infection. 293T

cells were either left untreated, transfected with Tim-1 and the support plasmids NP, VP30,

VP35, but not L, or the mini-genome plus all the support plasmids. (A) Polygonogram of 28 two-

drug combinations (at monotherapy IC50 doses) administered at 24 hrs post-trVLP infection, then

luciferase activity evaluated 3 days later. A thick red line represents strong synergy between two

drugs (CI<1), a thin black line represents additive effects (CI=1), and a thick blue line represents

strong sub-additive (less than additive) between two drugs (CI>1). (B-I) Polygonograms of 56

three-drug combinations. The backbone drug in each triple combination is listed above the

heptagon, and the synergism/sub-additive effect of the additional two drugs is represented within

each heptagon. (J-K) Combination index (CI) vs. fractional inhibition (Fi, percent luciferase

inhibition) plots of the most synergistic and sub-additive double (J) and triple (K) drug

combinations on trVLP luciferase activity. Dotted lines identify thresholds of synergy/additive

effects. Values shown are the means of 2-4 biological replicates in 2 independent experiments.

Error bars are the standard error of the mean.

164

Table 4.1: Fi and CI values for two-drug combination treatments. Experimental details are

as described in the legend to Figure 4.8. The fractional inhibition (percent luciferase inhibition),

combination index and strength of synergy (+), additive effects (+/-) or sub-additive (-) of each 2

drug combination therapy are shown.

165

Table 4.2: Fi and CI values for three-drug combination treatments. Experimental details are

as described in the legend to Figure 4.8. The fractional inhibition (percent luciferase inhibition),

combination index and strength of synergy (+), additive effects (+/-) or sub-additive (-) of each 3

drug combination therapy are shown.

166

In a final series of experiments, in order to validate our findings from the trVLP infection

studies, we examined the antiviral effectiveness of IFN-ß, IFN-α, TOR, FVP, AZT, 3TC and

TFV in 293T cells infected with ZEBOV (ZEBOV contained an eGFP reporter). CDV was

excluded from these experiments. Initial dose-response studies were conducted at doses

reflective of those used in the trVLP experiments in Figure 4.4. A higher dose of each drug was

required to inhibit ZEBOV infection compared with trVLP infection (Fig. 4.9). Using the IC25 of

each drug, we next evaluated 2 and 3 drug combinations for additive or synergistic effects

against ZEBOV infection. All seven 2 drug combinations were synergistic (low CI) (Fig,

4.10A), similar to the most synergistic combinations against trVLP in Figure 4.8J. IFN-β + 3TC

proved to be the most synergistic 2 drug combination, analogueous to trVLP infection. Of the

most synergistic 3 drug combinations identified in the trVLP infection system, all seven

exhibited synergy against ZEBOV infection, with IFN-β + 3TC + AZT and IFN-β + TOR + AZT

exhibiting the strongest synergy (Figure 4.10B). The CIs determined from trVLP infection

correlated well with those determined using ZEBOV infection; specifically, the correlation

coefficients (R2 values) confirm this (Figure 4.10C-D).

167

Fig. 4.9: IFNs, toremifene and nucleoside analogues administered 24 hrs post-exposure

inhibit ZEBOV-GFP. (A-H) 293T cells were infected with ZEBOV-GFP (MOI = 0.1).

Twenty-four hours post-infection cells were either left untreated, or treated with the indicated

drugs at the indicated doses. Intracellular GFP was measured 48 hours later and the percent

inhibition quantified relative to infected, untreated cells (DMSO solvent control). Values are the

means of 4 biological replicates. Error bars are the standard error of the mean.

168

Fig. 4.10: Synergistic 2 and 3 drug combinations against trVLP-LUC infection inhibit fully

infectious ZEBOV-GFP. (A-B) 293T cells were infected with ZEBOV-eGFP at an MOI of 0.1,

then treated with 2 and 3 drug combinations as indicated, 24 hours post-infection, at their

monotherapy IC25 doses. GFP fluorescence was measured 3 days post-infection. Values are the

means of 4 biological replicates, and error bars represent the standard error of the mean. Data

are representative of 2 independent experiments. The combination index (CI) was plotted

against the fractional inhibition (Fi, percent GFP inhibition) for each drug combination. (C-D)

Plot of CIs for ZEBOV infections compared with CIs for trVLP infections.

169

4.5 Discussion

In September 2014, the WHO hosted a conference to facilitate development of a global action

plan to deal with the Ebola outbreak in West Africa. Delegates from affected West African

countries, ethicists, scientists, health care providers, logisticians and representatives from

different funding agencies were in attendance. A committee had been struck to evaluate the

different vaccine candidates and therapeutic interventions being developed, which subsequently

received an overwhelming number of submissions for consideration, and was hampered by an

inability to compare antiviral effectiveness, since in vitro and pre-clinical in vivo model systems

vary, treatment regimens vary from prophylaxis to post-exposure administration, and direct

readouts of antiviral efficacy differ. Moreover, given the virulence and high mortality associated

with EVD, all of these studies have been conducted under BSL 4 conditions, limiting the number

of laboratories that can engage in these antiviral studies. Cognizant of these limitations, we

employed the trVLP model system to compare the antiviral effectiveness of eight antiviral

candidates from three drug classes. We evaluated their antiviral activities in the context of

inhibition of Ebola replication, using this mini-genome model that allows for rapid comparisons

among compounds under BSL 2 conditions. The tetracistronic minigenome represents the most

sophisticated in vitro replication model of Ebola virus to date. trVLPs proceed through every

replication step as wild type Ebola virus, and have been tested in multiple cell lines. Using TOR,

there has been some validation of the trVLP assay. Specifically, TOR has been evaluated in

limiting Ebola virus infection of VeroE6 and HepG2 cells, and exhibited IC50 values of 0.2 µM

and 0.03 µM, respectively [586], in line with the IC50 dose for TOR (0.36 µM) observed with

trVLP infection. Likewise, the IC50 identified in the trVLP system for FPV (36.8 µM), is

consistent with that of 67 µM recorded using Ebola virus infection [23], suggesting that this

Ebola mini-genome system has relevance for screening potential antiviral compounds. Indeed,

our validation studies using ZEBOV (ZEBOV-eGFP) suggest that the trVLP infection model has

utility as an in vitro screening assay when comparing different drugs as monotherapies or in 2

and 3 drug combinations.

170

As mentioned, the Ebola virus encodes in its genome factors that limit a type I IFN response to

infection [577-579]. Yet, both rodent and non-human primate studies suggest that IFN-α and

IFN-ß treatment can confer partial protection from infection, reducing viremia and prolonging

survival [31, 580, 581], suggesting that it may be possible to override the inhibitory effects of the

virus by treatment with IFN. At the outset, we conducted a series of experiments to compare the

antiviral activities of IFN-α and IFN-ß in the trVLP infection system, and our findings suggest

that whether treatment is administered prior to or post-infection, both IFN-α and IFN-ß exhibit

antiviral activity. These findings only have relevance for the direct antiviral activities of these

IFNs, since the effects of IFN-α or IFN-ß on immune modulation for viral clearance cannot be

determined using this system. Nevertheless, these data contributed to the decision to conduct a

clinical trial of IFN-ß treatment for EVD in Guinea.

We provide evidence that the nucleoside/nucleotide analogues 3TC, AZT, TFV, FPV and CDV

inhibit Ebola trVLP replication in vitro. The results with 3TC are in contrast to published data

that show no evidence for 3TC inhibiting Ebola virus infection in vitro [519]. These studies

examined the antiviral effectiveness of 3TC when administered one hour prior to infection, in

contrast to our studies that have focused on post-exposure protection. In cells, the kinetics of

3TC phosphorylation are such that a minimum of four hours are required for optimal activity,

perhaps distinguishing why our 24 hour pre-treatment, specifically a combination treatment,

offered protection. Post-exposure treatment with 3TC and the other nucleoside/nucleotide

analogues we examined, would more likely reveal activity against viral RNA synthesis than pre-

treatment. When comparing the IC50 values of each of the nucleoside analogues that we tested,

TFV exhibited the lowest IC50 at ~1 µM. Whether this reflects the fact that this adenosine

monophosphate analogue only requires two phosphorylation events to become an active drug

versus three for the other nucleoside analogues, remains undetermined. Extensive published data

reveal both the safety profiles [523, 587, 588] and the biodistribution of 3TC, AZT and TFV in

the circulation and liver [416, 538], the same compartments where Ebola infects monocytes,

macrophages, dendritic cells, endothelial cells and hepatocytes. Moreover, drug interactions

with other nucleoside analogues have been well studied: e.g. tenofovir disoproxil fumarate, when

171

used alone or in combination with emtricitabine effectively prevents HIV-1 infection in

antiretroviral pre-exposure prophylaxis (PrEP) [588].

Our studies also revealed that the active metabolite of brincidofovir, CDV, has a narrow

therapeutic window of efficacy (6.25-25 µM) when assessed in the trVLP assay, enhancing viral

replication at higher doses when added either prior to or post-infection. In cell viability assays,

CDV exhibits cytotoxicity at doses >10 µM. These findings suggest that caution is required if

CDV is to be considered further for the treatment of EVD, specifically that phase I/II trials

define the safety profile of this drug for EVD.

Another advantage of this in vitro system is that it allowed us to evaluate various 2 and 3 drug

combinations and demonstrates that combination treatments limit viral replication up to 97.3%.

A benefit of combination treatment is the potential to limit/avoid the emergence of drug

resistance. Interestingly, IFN-ß was predominant among all the 8 antivirals considered in terms

of contributing very strong synergism in combination treatments: e.g. IFN-ß + 3TC; IFN-ß +

3TC + AZT. Using this system, we observe that FPV, when administered 24 hours post-

infection, has an IC50 of ~ 37 µM. To date, the phase II/III JIKI trial examining the efficacy of

FPV against EVD has reported only modestly encouraging results. In our 2 drug combination

treatment studies we show that, with the exception of CDV, whenever FPV is included, synergy

occurs, effectively reducing the CI. It may transpire that for treating EVD, FPV is most effective

in a drug combination regimen.

Viewed altogether, we present an in vitro Ebola trVLP screening system, that requires only level

2 biocontainment, which allowed us to compare the antiviral activities of 8 compounds, either

alone or in combination. We provide evidence that IFNs are effective inhibitors of Ebola

replication, with IFN-ß exhibiting greater efficacy over IFN-α, or when used in combination with

nucleoside analogues. We infer from our data that whether IFN-ß treatment is administered 24

hours prior to, or up to 24 hours post-infection, reduced Ebola replication is achieved. As

additional antiviral therapeutic candidates become available, we now have the capability to

measure and compare their direct antiviral activities with the existing panel. This allows for rapid

172

in vitro evaluation and the opportunity to prioritize antiviral candidates for further pre-clinical

and clinical trial studies.

173

Chapter 5: Key Findings, Future Perspectives,

Conclusions and Broader Significance

174

5.1 Key Findings, Limitations and Future Perspectives

HIV-1

As new indications for small-molecule kinase inhibitors expand beyond the treatment of various

cancers [169, 170], the essential role of non-receptor tyrosine kinases facilitating viral replication

are increasingly being recognized, along with their associated disease pathologies that can be

targeted with small-molecule inhibitors [146, 178, 201, 554]. At the outset, this work

investigated the role of the SFK member c-SRC during early HIV-1 infection, to determine

whether it has particular signaling functions during early HIV-1 replication in diverse CD4+

T-

cell lines and primary human CD4+ T-cells. A variety of techniques were used, including SFK

tool-drug compounds PP1, PP2 and SU6656, ADV vector overexpressing DN c-SRC, and

targeted siRNA knockdown (chapter 2). It was also determined whether the focal adhesion

kinase binding partner PTK2B exhibited similar affects as c-SRC during early HIV-1 infection,

and a variety of HIV-1 viruses were considered, including: X4- or R5-tropic viruses, nef-

deficient or fully infectious viruses, and laboratory strains versus primary clinical isolates. The

essential role of c-SRC during early time-points of HIV-1 infection led to further exploration of

four potent c-SRC kinase inhibitors, providing evidence to support further testing of these

preclinical or FDA-approved compounds for further studies of HIV-1 inhibition (chapter 3).

Lastly, the work presented in this thesis expanded on the use of FDA-approved drugs, used to

treat HIV-1, for repurposing for use in EVD. Monotherapy and combination antiviral therapy of

8 antiviral candidates for inhibition of a ZEBOV mini-genome model of replication or in vitro

ZEBOV infection were used (chapter 4). Nucleoside analogues that were used for

compassionate EVD treatment during the 2014-16 ZEBOV outbreak in West Africa were a

particular focus [519]. The trVLP model of ZEBOV replication permitted rapid assessment of

drugs and combinations of therapies to prioritize testing of compounds, leading to confirmatory

BLS4 testing of the most synergistic drug combinations, and providing rationale for future drug

development in small animal models of EVD.

Prior to this work, the roles of LCK and FYN signaling had been the most studied in HIV-1

infection of CD4+ T-cells [227, 238], but an understanding of a role for c-SRC signaling had not

175

been clearly defined. Early LCK activation downstream of CD4 can be decoupled from the

TCR-CD3 complex by Nef and impair formation of the immunological synapse [589]. LCK also

becomes recycled with CD4 at endosomes, contributes to Tat-mediated HIV-1 transcription,

facilitates Gag-assembly during viral egress, and promotes virological synapse transmission of

HIV-1 [222, 226-228, 589]. Activated FYN signaling promotes HIV-1 transcription through

NF-κB, enhances assembly and release of virions, and yet becomes inhibited by viral Vif to

reduce phosphorylated APOBEC3G packaged into budding virions [227, 237, 238]. Similar to

both LCK and FYN, this work demonstrates that c-SRC becomes activated and phosphorylated

at Tyr419 within the first hour of HIV-1 IIIB (X4) or HXB2 (X4) infection of Jurkat C T-cells

(chapter 2), or HXB2 (X4) or JR-FL (R5) infection of activated primary CD4+ T-cells (chapter

3). This was strongest for virus expressing Nef, but not completely absent in pseudo-enveloped

viruses lacking Nef (HXB2 or JR-FL), which may suggest c-SRC activation is partially

independent of Nef in the first hour of CD4+ T-cell infection. Similar results were found for the

binding partner PTK2B, where its phosphorylation at Tyr402 increased after 1 hr of HIV-1 IIIB

infection in Jurkat C T-cells, or HXB2 or JR-FL infection in activated CD4+ T-cells. However,

PTK2B was not phosphorylated at Tyr402 after 1 hr of HXB2 infection in Jurkat C cells. This

was the first of many indications that signaling within T-cell lines may not be the best models to

recapitulate physiological signaling cascades of c-SRC or PTK2B in primary CD4+ T-cells.

In chapter 2, it was demonstrated that pan-inhibition of SFKs with 10-20 μM of the tool

compound SU6656 significantly enhanced HIV-1 luciferase activity of nef-deficient VSV-

G/HIV-1 in Jurkat C or Jurkat E6-1 infection. This observation correlated with decreased in

vitro c-SRC kinase activity and no change in cell viability. The SU6656 experiments were

corroborated with follow-up tests of DN c-SRC ADV vector overexpression, which increased

VSV-G/HIV-1 infection in Hut 78 and Kit 225 cells. However, these ADV results should be

interpreted cautiously, as ADV treatment alone caused high cell death in Jurkat E6-1 and Hut 78

cells (70-80% of cells were 7-AAD+), and EYFP transfection efficiency was not equivalent

between cell lines. These findings suggest further optimization of ADV vector MOI is needed in

the three T-cell lines. Future experiments should also evaluate whether a dominant positive (DP)

mutant of c-SRC decreases VSV-G/HIV-1 infection in T-cell lines, as would be predicted. This

176

work from chapter 2 also revealed that siRNA reduction of c-SRC or PTK2B expression in

Jurkat E6-1 cells increased HXB2 reverse transcription. The pools of 4 siRNAs were kinase-

specific, had no affect on cell viability when administered by lipofection, nor change in surface

expression of CD4 or CXCR4 (chapter 2). The increased HXB2 luciferase activity from c-SRC

or PTK2B siRNA knockdown also correlated with increased early and late reverse transcript

cDNA, as well as 3-4-fold higher integrated virus after 24 hrs of infection.

However, these drug and siRNA findings are in contrast with the results of primary CD4+ T-cells

studied in chapter 3, and the published research of others describing a protective effect of c-SRC

inhibition or PTK2B siRNA knockdown 6 days post-infection [264, 345]. Different SFK

inhibitors were employed in chapters 2 and 3 that inhibit different off-target kinases. SRC

Inhibitor-1 is reported to be the most selective for SFKs at 1 μM in an in vitro study of 73 human

kinases (4 off-targets), followed by PP1 (5 off-targets), PP2 (5 off-targets) and SU6656 (9 off-

targets) [557]. In comparison, dasatinib, saracatinib and KX2 391 are less selective inhibitors of

SFKs, targeting several other kinases such as ABL1, ARG and serine/threonine kinases [590,

591]. Yet employing the same pool of siRNA to knockdown c-SRC in chapters 2 and 3 caused

opposite changes to luciferase activity in HIV-1 infected Jurkat cells compared with primary

CD4+ T-cells, suggesting the observed differences are primarly caused by the cell type

investigated.

Taken together, these findings cast doubt on the generalizability of c-SRC and PTK2B signaling

in transformed T-cell lines, with their corresponding signaling events in primary human CD4+ T-

cells during HIV-1 infection. Indeed, a comprehensive study on the proximal TCR signaling in

Jurkat E6-1 and Hut 78 cells, relative to activated primary CD4+

T-cells, showed that upon TCR

stimulation the cell lines exhibited greater Ca2+

flux, uncharacteristic expression of costimulatory

receptors, atypical cytokine release, and hyperphosphorylation of PTK2B and c-SRC, among

other downstream signal transduction abnormalities (see Figure 5.1, originally published in

[562]). Thus, while mutant T-cell sublines can be useful in other contexts [592], the work in this

thesis shifted instead to focus on the role of early c-SRC and PTK2B signaling in HIV-1

infection of primary CD4+ T-cells, isolated from healthy human donors (chapter 3).

177

Fig. 5.1: Aberrant TCR signaling in Jurkat T-cells. PTK2B (Pyk2) and other proximal

signaling kinases are hyperphosphorylated in Jurkat E6-1 compared with primary activated CD4+

T-cells. Overexpression or hyperphosphorylation are green, while underexpression or

hypophosphorylation are in red [562].

178

In activated primary CD4+

T-cells, this work demonstrates that c-SRC co-immunoprecipitates

with a truncated PTK2B fragment phosphorylated at Tyr402, 1 hr following infection of nef-

deficient HXB2 or JR-FL virus (chapter 3). To explain this finding, HIV-1 chemokine receptor

binding may induce cleavage of PTK2B, potentially through CXCR4, CCR5, or P2Y2 activation

and signaling [147, 345]. This highlights a potential PTK2B similarity with closely related FAK

during HIV-1 infection. Downstream of gp120/CD4 signaling, phosphorylated FAK becomes

cleaved by caspase-3 and caspase-6 into a 90 kDa fragment containing the FAT domain, and a

35 kDa fragment containing the kinase domain of FAK [322, 346]. Separating the FAT domain

from focal adhesions in this manner has been shown to promote p53-dependent apoptosis [347,

348]. Thus, it should be explored in future experiments whether during HIV-1 infection similar

signaling from the larger PTK2B fragment containing the FAT domain occurs, as the smaller,

phosphorylated kinase fragment of PTK2B binds to c-SRC.

To investigate the effects of activated c-SRC and PTK2B on the early stages of HIV-1

replication, careful optimization of siRNA electroporation was performed, as CD4+ T-cells are

challenging non-adherent cells to transfect (chapter 3). Moreover, activated primary CD4+ T-

cells degrade argonaute, an enzyme essential to the RNA-Induced Silencing Complex (RISC)

[593]. Without argonaute, many mRNAs become de-repressed, which promotes rapid protein

translation as part of normal T-cell activation and T-cell differentiation [593]. This natural

phenomenon of argonaute degradation upon activation therefore precluded testing of c-SRC or

PTK2B siRNA knockdown after T-cell activation. Primary CD4+

T-cells administered siRNA,

and then activated, showed no changes to CD4, CXCR4 or CCR5 expression, or reduction in cell

viability (chapter 3).

The findings in chapter 3 reveal that viral reverse transcription of either HXB2 or JR-FL was

unaffected by c-SRC or PTK2B siRNA knockdown. However, reducing c-SRC expression in

CD4+ T-cells prior to infection with nef-deficient HXB2 or JR-FL viruses, increased the

detection of early reverse transcripts, late reverse transcripts, and 2-LTR circles, while reducing

viral integration and luciferase reporter activity. Similar results were found after PTK2B siRNA

knockdown, although only during JR-FL (R5) infection. This suggests PTK2B activation is a

byproduct of HXB2 (X4) and IIIB (X4) entry, perhaps through CXCR4 stimulation [147], and is

179

not essential in the early stages of HXB2 or IIIB replication in an activated CD4+ T-cell. While

both c-SRC and PTK2B are activated upon HIV-1 binding and fusion, they could be transmitting

signals that prepare the cell for later stages of the intracellular virus replication cycle [147]. To

generalize these c-SRC and PTK2B findings to multiple strains of HIV-1, testing of more X4 and

R5 HIV-1 clinical isolates are needed. Nonetheless, infection with HIV-1 strains expressing Nef

(IIIB (X4), Ba-L (R5) or KNH1207 (R5)) could not rescue infection after c-SRC siRNA

knockdown, as quantified by qPCR integration and p24 ELISA measurements, suggesting the

function of c-SRC may be independent of Nef during early infection. This outcome was not

entirely surprising, given the low affinity of in vitro c-SRC SH3 binding to Nef PxxP, relative to

the stronger binding of HCK or LYN to Nef that is mediated by a key Ile residue in their SH3

domain [164]. Collectively, these results from chapter 3 suggest changing roles for c-SRC and

PTK2B signaling at various time-points and stages of the early HIV-1 replication cycle, similar

to SFKs LCK and HCK during HIV-1 infection [223, 227, 238, 249]. In future experiments, it

will be of interest to test whether c-SRC or PTK2B siRNA knockdown increase HIV-1 reverse

transcripts and reduce viral integration in a similar fashion in T-cell sub populations. These

experiments could be performed in T-cells that maintain the viral reservoir in lymph nodes

(central memory peripheral T-follicular cells), prevent microbial translocation at the gut and

control inflammation at mucosal barriers (Th17 cells), or play a role in suppressing immune

hyperactivation (Tregs) [80, 594, 595].

The increase in observed early and late reverse transcripts of JR-FL virus after c-SRC or PTK2B

siRNA knockdown, together with no effect on RT activity, strongly suggest these kinases act

after reverse transcription and prior to viral integration (chapter 3). Other kinases have shown

similar effects on HIV-1, such as JNK inhibition increasing reverse transcripts and impairing

viral integration [159]. Unlike LCK and FYN, c-SRC interacts with membrane proteins of

multiple compartments in addition to microdomains at the plasma membrane [189], suggesting

c-SRC could have roles in the cytosolic or nuclear stages of HIV-1 replication. Moreover, the

SH3 domain of c-SRC regulates its localization from actin-associated focal adhesions to the

perinuclear region in a microtubule-dependent manner [189]. The function of host and viral

proteins that constitute the pre-integration complex of HIV-1 are still under investigation [155].

180

It also remains to be determined whether c-SRC or PTK2B directly phosphorylate host and viral

proteins in the PIC, or bind PIC proteins through SH2 or SH3 interactions to stabilize the

complex. In support of these hypotheses, c-SRC has been predicted by NetPhos 3.1 to

phosphorylate HIV-1 RT at eight different sites in its protein sequence (Tyr56, Tyr115, Tyr188,

Tyr342, Tyr354, Tyr405, Tyr441 and Tyr457) with 31-52% confidence, and three sites in the IN

sequence (Tyr15, Tyr83 and Tyr99) with 37-48% confidence [596]. Comparing with dengue

virus infection, it has been proposed the related family member FYN phosphorylates host factors

in that RNA replication complex, or may act as an adaptor protein that scaffolds interactions

between components of the dengue virus replication complex [178]. In addition, c-SRC has been

shown to directly interact with the HCV RdRP complex during HCV infection, facilitating

intracellular replication [202]. In the context of HCV replication, the SH3 domain of c-SRC is

exploited to bind the viral polymerase NS5B, linking c-SRC to the non-structural phosphoprotein

NS5A through its SH2 domain [202]. These c-SRC interactions were also found to be essential

for NS5B and NS5A binding each other and HCV replication, and were not mediated by related

SFK members FYN or c-YES [202]. Thus, c-SRC could have a novel and essential interaction

within the PIC of HIV-1. This line of inquiry is part of future experiments that involve isolating

the HIV-1 PIC, to determine a potential scaffolding role for c-SRC.

The buildup of 2-LTR circles and reduced proviral integration after c-SRC or PTK2B

knockdown further implicate these kinases in the nuclear translocation of the HIV-1 PIC (chapter

3). Indeed, this work showed that in the presence of the integrase inhibitor raltegravir, the

synthesis of HXB2 or Ba-L cDNA was similar to the nuclear effects of c-SRC or PTK2B siRNA

knockdown. Others have reported similar increases in 2-LTR circles after raltegravir

intensification in HIV-1 patients taking cART [565]. There is precedent for non-receptor kinases

affecting the nuclear biology of HIV-1, such as MAPK phosphorylation of the inner nuclear

lamina protein emerin (EMD) [154]. If EMD phosphorylation is blocked, or EMD expression

silenced by siRNA, proviral integration decreases and 1- and 2-LTR circles accumulate [154].

The phosphorylation of nuclear proteins may very well be a common lentivirus mechanism for

accessing the nuclear compartment [154], and may include c-SRC kinase activity during HIV-1

infection. c-SRC has been found to localize to the nucleus and nucleolus, and may regulate

181

proteins involved in cell cycle progression and entry into mitosis [189]. Tyr419 phosphorylation

on nuclear c-SRC has been shown to induce c-SRC interactions with Sam68 in other cell types

[597]. Thus nuclear c-SRC may already have a role in HIV-1 replication through Sam68, as

Sam68 regulates HIV-1 RNA processing, the transactivation of Rev, viral mRNA nuclear

transport, and mRNA translation in the cytosol [317]. Research into c-SRC protein interactions

with perinuclear membrane and nuclear protein substrates is ongoing [189]. One hypothesis is

that nuclear c-SRC could be regulating DNA ligase 4 (DNAL4), an essential DNA repair protein

in the NHEJ pathway that specifically catalyzes the ligation of unintegrated HIV-1 cDNA into 2-

LTR circles [155]. It has been put forth that the free ends of unintegrated HIV-1 cDNA are

sensed as DNA double-strand breaks, which activate nuclear kinases that regulate DNA repair

mechanisms to circularize viral cDNA, thus pre-empting apoptosis signaling by the T-cell [155].

Our lab is now investigating the potential role of c-SRC signaling in this NHEJ repair pathway

that circularizes HIV-1 cDNA into 2-LTR circles during HIV-1 replication [598].

The findings in chapter 3 also demonstrate the novelty of four specific c-SRC inhibitors that

strongly reduce JR-FL or HXB2 infection in activated CD4+ T-cells. Dasatinib, saracatinib,

KX2-391 and SRC inhibitor-1 all inhibited the luciferase activity of these recombinant viruses,

and caused little to no increase in apoptosis or necrosis. Dasatinib was the most potent of the

four inhibitors examined, reducing HXB2 or JR-FL infection in the 10-100 nM range. This drug

has recently been shown to inhibit the phosphorylation of SAMHD1, a c-SRC substrate [537].

Without phosphorylation of negative regulatory tyrosine residue Tyr592, SAMHD1 becomes

activated and reduces the cytosolic pool of free dNTPs, impairing HIV-1 reverse transcription in

dasatinib-pretreated primary CD4+ T-cells [537]. Dasatinib is a safe and well tolerated kinase

inhibitor, approved for second-line treatment of CML [553]. Moreover, the related SRC/ABL

inhibitor imatinib has been used to safely treat KS or CML cancers in HIV-1 patients on fully

suppressive cART, improving long-term survival [176, 599, 600]. These clinical examples of

kinase inhibitor provokes the question: Can dasatinib be used safely in conjunction with cART to

further suppress HIV-1 infection? At doses within well-tolerated levels in humans, dasatinib has

also been shown to inhibit the ex vivo reactivation of HIV-1 from the CD4+

T-cells of treatment

naïve, HIV-1 donors [554]. To investigate the mechanism of action of dasatinib, which targets

182

Abelson murine Leukemia viral oncogene homolog 1 (ABL1) and Abelson-Related Gene (ARG)

kinases as well as c-SRC, the role of these two ABL family kinases during HIV-1 infection is

another active area of future research. Furthermore, current investigations in our lab are

expanding on the findings presented in chapter 3, to test whether dasatinib treatment can reduce

in vivo Ba-L infection in a humanized mouse model of HIV-1 infection. This model will be an

excellent means of studying HIV-1 resistance mutations that may develop from weeks of

dasatinib monotherapy, which could be pre-empted with combination therapy if they occur. We

will also explore whether dasatinib can be included in cART as an alternative integration

inhibitor in this mouse model of HIV-1 infection. Furthermore, CRISPR/Cas9 may be used to

knockout c-SRC or PTK2B in human CD34+

Hematopoietic Progenitor Cells (HPCs) prior to

their transplantation into mice, to ascribe more definitive in vivo roles of c-SRC and PTK2B

signaling during HIV-1 infection.

ZEBOV

In September of 2014, during the height of the ZEBOV outbreak in West Africa and when the

first documented case of ZEBOV transmission occurred in the United States [601], the need to

rapidly assess drugs for preclinical ZEBOV antiviral activity was of paramount concern to the

National Microbiology Laboratory of Canada, US CDC, USAMRIID, and the WHO Working

Group on Ebola Therapeutics. By establishing a new trVLP model of ZEBOV replication in the

lab [534], the four c-SRC inhibitors from chapter 3 were initially tested for activity against

ZEBOV trVLP replication. While this SFK research was a novel avenue of its own, and

preliminary results were promising, the urgent need to test and compare experimental therapies,

either fast-tracked for upcoming phase II/III clinical trials in West Africa (BCV, FPV and IFN-

β), or already being used for compassionate EVD treatment (AZT and 3TC), took precedent for

in vitro testing [29, 31, 431, 519]. Thus, 8 compounds from 3 drug classes were evaluated alone

or in combination, to provide additional rationale for further preclinical development or their

potential use in emergency ZEBOV clinical trials (chapter 4).

183

HEK 293T cells are an ideal cell line for the ZEBOV trVLP model because their high

transfection rate produces many virions [534], and the cells can quickly convert nucleoside

analogs to their active triphosphate form analagous to primary hepatocytes [418]. Indeed,

VeroE6 show slower phosphorylation of nucleoside analogs and may under represent drug

potency [418]. As 293T cells are not the natural target cell infected by ZEBOV, it was important

to first validate the in vitro model with therapeutic treatments published to inhibit fully infectious

ZEBOV in other cell lines [23, 586]. The ZEBOV GP1,2 entry inhibitor TOR showed very

similar IC50 results in the trVLP model (0.36 μM) as with publications of TOR inhibiting

ZEBOV in VeroE6 (0.2 μM) or HepG2 cells (0.03 μM) [586]. Similarly, FPV has been reported

to inhibit ZEBOV in VeroE6 cells with an IC50 of 67 μM [23], comparable to the IC50 of

36.8 μM found for ZEBOV trVLP in chapter 4. Together, this data suggested the trVLP model

of ZEBOV infection in 293T cells could be used to screen potential antiviral compounds.

As predicted from in silico simulations of RdRP L nucleotide analogue docking [456], 3TC,

AZT and TFV each showed dose-dependent inhibition of ZEBOV trVLP luciferase activity,

which was independent of whether the drug was administered a day before, during or one day

after infection (chapter 4). TFV, which was predicted out of 20 nucleoside and nucleotide

analogs to have the strongest interaction with the putative RdRP L nucleotide binding pocket

[456], showed the lowest IC50 of the 5 nucleoside analogs tested (0.98 μM). This was followed

by AZT (4.2 μM), 3TC (6.2 μM) and CDV (7.7 μM) giving similar doses for 50% inhibition.

Interestingly FPV had the highest IC50 (36.8 μM). FPV is a drug that is required in high doses to

protect small animals from lethal ZEBOV exposure [29]. It is also required in high doses in non-

human primates to achieve target plasma levels [441]. Furthermore, FPV showed sub-optimal

dosing in the blood samples of EVD patients in the recently completed phase II JIKI trial in

Guinea [444]. From these multiple lines of investigation, it would appear FPV is a mild inhibitor

of ZEBOV RdRP that could benefit from synergistic drug combinations to reduce the high doses

of drug required in monotherapy.

The work presented in chapter 4 is the first account of 3TC, AZT or TFV having antiviral

activity against ZEBOV trVLP replication or against ZEBOV-GFP in vitro infection in BSL4.

As a potential mechanism of action, these three nucleoside/nucleotide analogues were then

184

shown to inhibit viral synthesis of negative-sense genomic vRNA and positive-sense cRNA and

mRNA, as measured by qRT-PCR. TFV showed the strongest antiviral activity of the 5

nucleoside analogues tested in this qRT-PCR assay. At first, these findings appear to be in

contrast with recent published work suggesting 3TC and AZT exhibit weak in vitro activity

against ZEBOV replication [518]. However, the publication showed similarly mild

dose/response curves of 3TC and AZT against ZEBOV-GFP, as reported in chapter 4. For

instance, 25 μM of 3TC caused 18% inhibition of ZEBOV-Makona in their report [518], while

the same 25 μM dose of 3TC caused 22% inhibition of ZEBOV-GFP in chapter 4. It was

anticipated that the required drug doses would be higher when moving from the ZEBOV trVLP

model to fully intact ZEBOV-GFP infection at an MOI of 0.1. Nevertheless, the primary value

in testing 3TC and AZT came not from monotherapy alone, but their potential to synergize in the

trVLP model of ZEBOV infection. In the top 7 two-drug and top 7 three-drug combinations

demonstrating the strongest synergy against ZEBOV-GFP, 3TC was included in 5 of these

combinations, and AZT was in 5 combinations as well (chapter 4). Likewise, the recent ZEBOV

study testing 3TC and AZT also reported drug synergy when combining 12.5 μM of 3TC with 25

μM of AZT, markedly reducing ZEBOV-Makona infection by 70% [518]. To further clarify the

mechanisms of 3TC and AZT ZEBOV inhibition, future work demonstrating RNA chain

termination of RdRP L in the presence of either nucleoside analogue, and crystal structures of the

RdRP L enzyme bound with either compound, would be desirable. In addition, the antiviral

activity of 3TC and AZT in ZEBOV infection should be confirmed in primary monocyte-derived

macrophages, to strengthen the preclinical evidence for evaluating these two drugs further in

small animal models of ZEBOV infection.

Treating 293T cells with the metabolite of BCV (CDV) at or above 100 μM prior to or post-

ZEBOV trVLP infection, unexpectedly and consistently increased viral luciferase activity 4 days

post-infection (chapter 4). Although CDV inhibited trVLP luciferase activity in a small range of

concentrations (1.25-25 μM), it reduced the antiviral activity of many of the other 7 drugs tested

in two- or three-drug combinations. Others have reported BCV to enhance ZEBOV-GFP

infection in Huh7 cells, at doses less than 0.2 μM [28]. In two recent phase III trials, BCV has

failed to show efficacy in treating ADV infections or preventing CMV infection during

185

hematopoietic cell transplantation in patients. Additionally, a third phase III trial of BCV to

prevent CMV infection after kidney transplant was recently terminated. BCV was originally

created to be a less toxic alternative to CDV, and shows no associated nephrotoxicity in patients

[602]. However, an oral BCV dose of 200 mg twice a week in a study of 30 participants was

shown to elevate liver enzymes detected in blood samples, such as Alanine Transaminase (ALT)

(40% of participants were > 3x upper limit of normal range) [413]. Elevated bloodstream levels

of liver enzymes can indicate potential inflammation or injury to the liver [603]. Two hundred

mg of BCV twice a week also caused diarrhea for 70% of the participants, leading to 60% of

those taking the drug to withdraw from the study from adverse effects [413]. It is possible that in

the BCV phase II ZEBOV trial, which only recruited 4 EVD patients of which none survived

[24], BCV may have unintentionally exacerbated liver dysfunction and diarrhea symptoms.

Evidence supporting this are: 1) Patient #2 in the BCV ZEBOV trial had hepatic injury (high

ALT levels) and persistent diarrhea, leading to suspension of BCV treatment by day 7 [24]. 2) It

was determined in the JIKI trial, which measured baseline viremia in 58 EVD patients and serum

ALT over 25 days, that patients nearing death had elevated ALT levels compared with those

who survived [18]. Collectively, these BCV findings and CDV results presented in chapter 4

highlight the necessity of establishing clear preclinical in vitro antiviral activity and in vivo

animal efficacy in preventing or treating lethal ZEBOV challenge, prior to initiating a phase II

trial during an epidemic setting [24].

The last key finding from chapter 4 was that specific combinations of the 8 drugs tested

exhibited strong synergy against ZEBOV trVLP, which were confirmed with fully infectious

ZEBOV-GFP in BSL4. FPV showed synergy with all other drugs tested, suggesting

recombinant IFNs, TOR or nucleoside analogs could be used with FPV to reduce its

monotherapy dose and maintain the same level of inhibition. This is in line with FPV

combinations with ribavirin that synergistically inhibit other RNA viral infections, such as

(-)ssRNA Lassa virus, Pichinde virus and RVFV, and (+)ssRNA Junin arenavirus [435, 436,

530]. Moreover, synergistic two drug combinations, such as IFN-β + 3TC or IFN-β + AZT,

were predictive of the synergy when testing the three drugs together (IFN-β + 3TC + AZT). Of

combinations only comprising nucleoside/nucleotide analogues, FPV + TFV showed the highest

186

synergy. This result was predicted in silico from their combined interaction energy displacing

NTPs at the putative nucleotide binding motif of RdRP L, with FPV binding from the “left” and

TFV binding from the “right” of the polymerase nucleotide binding pocket (see Figure 5.2 for

binding of TFV + FPV outcompeting NTP binding, originally published in [456]). In addition,

the in vitro trVLP model in chapter 4 permitted rapid testing of multiple drug combinations, with

hits inhibiting luciferase activity as high as 97.3% and with no appreciable affect on cell

viability. This data allowed for 14 combinations to then be prioritized for BSL4 testing at the

National Microbiology Laboratory in Winnipeg. As in ZEBOV trVLP infection, the

combinations of IFN-β + 3TC or IFN-β + 3TC + AZT showed the strongest drug synergy against

ZEBOV-GFP in vitro. Moreover, the consistency in which low doses of recombinant IFN- β

inhibited ZEBOV alone or in combination added to the strong preclinical evidence supporting

this treatment to be considered in a phase II ZEBOV clinical trial [22, 31]. On March 26th

2015,

a phase II IFN- β-1a proof-of-concept, historically controlled, single-arm trial started in the

western town of Coyah, Guinea, and completed by June 12th

2015 [22]. While the results in

chapter 4 are but one of many sources of data that contributed to the planning of this trial, it is an

example of real-world impact this thesis has already made.

Presently, there are no active cases of acute EVD reported by the WHO in West Africa [5]. Yet

from May to June 2017, a small outbreak of EVD (5 confirmed cases, 3 probable cases) occurred

in the North-East province of Likati in the Democratic Republic of the Congo [604]. While the

WHO is confident this new outbreak will be contained, this should not engender research

complacency between larger EBOV outbreaks. On the contrary, this time provides an

opportunity for clinicians and scientists to produce rigorous and sound preclinical evidence that

better inform policy makers, who may again priorotize EBOV clinical trials during an active

EBOV outbreak. In our lab, future work has begun on screening antimalarial derivatives of

quinoline, to determine antiviral activity in the ZEBOV trVLP model of infection in 293T cells.

Moreover, earlier work on SFK inhibitors that inhibit trVLP replication has been

187

Fig. 5.2: Model of FPV-RTP and TFV-DP binding the RdRP L nucleotide pocket of

ZEBOV. Preferential binding of favipiravir-RTP (red) to the “left” side and tenofovir-DP

(orange) to the “right” side of the nucleotide binding motif (yellow). The total interaction energy

of both nucleotide analogues with the polymerase is significantly lower than each NTP (green),

displacing them to locations with decreased affinity. (A) ATP. (B) CTP. (C) GTP. (D) UTP

[456].

188

reinitiated. This investigation of host tyrosine kinases will likely be a promising new area of

research, as 53 host-protein interactions during ZEBOV infection of VeroE6 cells have recently

been described, and little is known of the role of NRTK signaling during ZEBOV replication

[357]. Only two such studies have been published [605, 606]. The first showed that ABL1

phosphorylation of VP40 matrix protein at Tyr13 was essential for budding of newly assembled

ZEBOV virions, by employing siRNA or a specific inhibitor (nilotinib) targeting ABL1 activity

[605]. The second publication also used FDA-approved kinase inhibitors (sunitinib and

erlotinib) to inhibit ZEBOV entry [606], providing additional rationale to explore SFK signaling

and the repurposing of kinase inhibitors in future ZEBOV lifecycle research.

189

5.2 Conclusions and Broader Significance

HIV-1

In summary, this work has specifically shown that c-SRC and PTK2B non-receptor tyrosine

kinases are key mediators of early HIV-1 replication in various T-cell lines and in activated

primary CD4+ T-cells from healthy donors. Using multiple experimental methods (including

tool-drug inhibitors, adenovirus vectors overexpressing DN c-SRC mutant, and targeted siRNA

knockdown) and testing of various strains of HIV-1 viruses (X4- or R5-tropic, fully infectious or

nef-deleted luciferase reporter viruses, VSV-G pseudo-enveloped viruses, laboratory strains and

primary clinical isolates), it was determined that reducing the activity or expression of c-SRC

consistently increased viral infection in transformed T-cell lines, but reduced viral infection in

primary CD4+ T-cells. Results with primary CD4

+ T-cells were taken as the more physiological

relevant results and further examined. Inhibition of viral infection of CD4+ T-cells was

associated with no change in RT activity, increased early and late reverse transcripts, reduced

proviral integration, and higher accumulation of 2-LTR circles, suggesting two novel

mechanisms by which this kinase may be altering intracellular HIV-1 replication: 1) At the level

of the pre-integration complex formation in the cytosol, and 2) During PIC nuclear translocation

and viral integration. Similar results were discovered for the c-SRC binding partner PTK2B.

However reduced PTK2B kinase expression only appeared to decrease HIV-1 infection when R5

viruses were tested. These findings offer the potential for better characterization of the PIC and

its associated host proteins, as arresting the virus at this stage from c-SRC siRNA knockdown

could allow for better isolation of this elusive protein/cDNA complex that is hard to characterize

in primary CD4+ T-cells [138, 607, 608].

The dependency of early HIV-1 infection on c-SRC signaling was a novel and complex finding.

This was unlike other SFKs FYN and LCK, which transmit signals as HIV-1 binds and fuses at

the plasma membrane, interact with viral Nef (LCK), change the recycling of surface proteins at

the trans-Golgi network (LCK), phosphorylate APOBEC3G (FYN) or facilitate viral egress

(LCK) [208, 217, 218, 223, 227, 238]. The work presented here strongly supports that c-SRC

has a unique cytosolic and nuclear role in the cell biology of HIV-1 infection of CD4+ T-cells. It

190

demonstrates that c-SRC regulates HIV-1 infection at multiple stages post-entry, reminiscent of

the integrase inhibitor raltegravir with respect to the nuclear effects of c-SRC [565, 566].

Furthermore, this research shows that four specific inhibitors of c-SRC (dasatinib, saracatinib,

KX2-391 and SRC Inhibitor-1), each with different off-target activity, can significantly reduce

HIV-1 infection of CD4+ T-cells. These four inhibitors, FDA-approved or under clinical

evaluation as potential cancer treatments [553, 561, 609], showed excellent cell viability data at

doses that significantly inhibit HIV-1 replication ex vivo. These results lay the foundation for

further preclinical testing of dasatinib, alone or concurrent with cART treatment, in a humanized

mouse model of HIV-1 infection.

The findings in this work make a significant advance in the fields of HIV-1 cell signalling and

novel HIV-1 treatment strategies, by demonstrating the roles of two non-receptor tyrosine

kinases during early HIV-1 entry of CD4+ T-cells, and evaluating kinase inhibitors that can

specifically inhibit one of these two targets. The fundamental roles of host kinases in the HIV-1

lifecycle are increasing being recognized, allowing for rapid discovery of kinase inhibitors as

new potential therapies of HIV-1 infection [146, 156, 160, 610]. Small-molecule kinase

inhibitors that can be repurposed for new indications in virology offer many significant

advantages [169, 170]. They often have low toxicity profiles, and broad tissue distribution. In

vitro studies suggests they may offer unique ways to inhibit HIV-1 replication in viral

sanctuaries [560, 611, 612], potentially altering the dynamics of the latent viral reservoir. They

can also be paired with latency reversal agents to prevent unwanted inflammation during “shock

and kill” methods [173]. Moreover, kinase inhibitors have the potential for ameliorating

underlining inflammation and neural cognitive dysfunction that occur during HIV-1 infection

[173, 613], which are not addressed adequately with current cART regimens. Kinase inhibitors

could also reduce the risk of cancers in HIV-1 patients, as demonstrated by their ability to treat

KS or CML without causing additional complications, extending the life expectancy of these

HIV-1 patients [176, 600]. Additionally, targeting host kinases essential for early intracellular

infection may pose higher barriers towards selecting drug-resistant mutations, as they would not

directly inhibit viral proteins [135, 159, 539]. Thus, tyrosine kinase inhibitors could be a useful

part of a multidrug regimen for those living with HIV-1. There is a strong need to design not

191

only intensifying therapies, but therapies that improve immune function in HIV-1 patients, such

as re-establishing CD4+ T-cell populations at gut mucosa [80]. The clinical application of SRC-

family kinase inhibitors of known specificity, biodistribution, pharmacokinetics, toxicity profile

and ease of administration, suggest these drugs could be repositioned as novel inhibitors of HIV-

1 infection, and should be explored further in small animal models of HIV-1 disease progression.

ZEBOV

The 2014-16 outbreak of ZEBOV in West Africa challenged global healthcare workers and

virology researchers from different backgrounds to come together and quickly assess which

preclinical drugs, treatments and vaccines should be prioritized for emergency evaluation in

phase II/III ZEBOV clinical trials. Many new molecules were discovered to have in vitro

antiviral activity [586], or improved the survival in animal models when treated after an

otherwise lethal ZEBOV exposure [6, 25]. The phase II/III ZEBOV clinical trials were the first

of their kind in an outbreak setting during a humanitarian crisis, which led to many lessons for

future trials, such as: ethical considerations of consent and the use of placebos; standardization of

intensive supportive care; the fostering of local, regional and global partnerships; establishing

multiple treatment sites as the spread of disease rapidly shifted; and new trial formats such as the

cluster-randomized ring design [16, 25, 26]. In particular, multiple approaches from HIV-1 drug

research, spanning from preclinical drug screening to treating patients who suffer not only from

EVD, but associated disease stigmatization, were very relevant in Liberia, Guinea and Sierra

Leone during the ZEBOV outbreak.

The creation of a sophisticated in vitro lifecycle model of ZEBOV in 2014 [534] allowed for

rapid assessment of 8 different drugs from three drug classes, including toremifene,

nucleoside/nucleotide analogues, and recombinant interferons. This thesis made significant

contributions by comparing and contrasting each of these compounds, testing various time-

points, doses, and 84 unique combinations, while assessing cell viability and antiviral activity

against a ZEBOV trVLP luciferase reporter virus. It was demonstrated for the first time that safe

and well tolerated nucleoside analogues 3TC, AZT and TFV, often used to treat HIV-1 or HBV

infection [408, 410], can inhibit ZEBOV trVLP replication in vitro. This was confirmed with

192

qRT-PCR measurements of ZEBOV vRNA and cRNA/mRNA synthesis 2 days post-infection.

The antiviral activity of 3TC, AZT and TFV were comparable or lower than the IC50 of

nucleoside analogues fast-tracked for phase II trials in Guinea and Liberia: FPV and the

metabolite of BCV (CDV) [18, 24]. While neither FPV nor BCV demonstrated significant

therapeutic benefit in these clinical trials, lessons were learned of unwanted side effects from

BCV administration [24], and lower than anticipated plasma levels from FPV monotherapy [18].

Retrospective analysis of compassionate FPV use suggest this drug should continue to be

developed for experimental treatment of EVD in a future EBOV outbreak, in addition to

intensive supportive care [445].

Testing 5 nucleoside/nucleotide analogs in the trVLP replication model confirmed many of the in

silico predictions of how these drugs might dock to the nucleotide binding motif of RdRP L and

outcompete NTP substrates [456]. TFV was predicted to have the strongest interaction energy

with the enzyme [456], and it was confirmed to have the lowest IC50 of the 5 nucleoside analogs

tested. Moreover, simulations of two nucleoside analogs flanking the nucleotide binding motif

from the “left” and “right” predicted strong synergy when combining TFV with FPV [456], a

drug combination that experimentally was confirmed to be highly synergistic in vitro. In

addition, testing of two-drug combinations rationally predicted synergistic three drug

combinations, adding more strength to the methodology used [533]. Lastly, confirmation of

antiviral activity of the 14 most synergistic drug combinations occurred with fully infectious

ZEBOV expressing a GFP reporter, demonstrating multiple efficacious combinations that

included FPV or IFN-β. Thus both of these drugs, which were evaluated in phase II ZEBOV

clinical trials as monotherapies [18, 22], could very well be improved by combinations with

other nucleoside analogues. These hypotheses should be investigated further in small animal or

non-human primate models of lethal ZEBOV challenge.

Combinations of monoclonal antibodies and siRNAs were behind the early success of ZMapp

and TKM-Ebola; however, they had shortcomings in drug production or treating people with

EVD in controlled clinical trials [27, 404]. Combinations with nucleoside analogues are now on

the leading edge of EBOV drug discovery, as ZMapp is actively being studied in combination

with either FPV or recombinant IFN-β. Thus, the nucleoside analogues 3TC, AZT and TFV

193

could fill the gap where monotherapy has so far failed to demonstrate therapeutic benefit, by

increasing the efficacy of other administered treatments as combination regimens. Moreover,

nucleoside analogues may have the additional advantage of exploiting the spontaneous mutation

rate of ZEBOV [614]. While the frequency of ZEBOV mutations are high, as other RNA

viruses, they are not represented in the genetic diversity of viable strains, suggesting nucleoside

analogues that increase the error rate of RdRP L cause harmful mutagenesis, reducing the fitness

of ZEBOV during infection [614]. Nucleoside analogs are also on the WHO list of essential

medicines, and can be deployed in resource limited settings, often without need of cold storage

[574]. Taken together, the findings presented in this thesis demonstrate the value of an in vitro

BSL2 trVLP screening system prior to BSL4 ZEBOV testing, allowing for direct comparisons of

8 compounds with suggested or known antiviral activity against ZEBOV replication [29, 31].

This work also contributes to the growing body of evidence advocating for combination therapy

as standard treatment of RNA viruses and retroviruses, to improve efficacy and theoretically

reduce the risk of drug resistance [456, 523, 530, 532, 615]. In future studies, this ZEBOV

trVLP model will continue to allow rapid assessment of new compounds of interest, such as SFK

inhibitors and quinoline derivatives, to directly compare their antiviral activity with leading

compounds in ongoing or planned ZEBOV clinical trials.

As of June 2016, there are approximately 17,300 Ebola survivors in West Africa, the largest such

Ebola cohort ever recorded [5]. The management of chronic disease that is becoming increasing

recognized as post-Ebola syndrome, is under active investigation [6, 450]. Moreover the

mechanisms of viral persistence, and the risk of ZEBOV reemergence and shedding, are also

being actively studied [357, 360]. Ebola survivors with persistent symptoms and low-level viral

replication in immune-privileged organs may benefit from antiviral drugs with appropriate

permeability, such as the nucleoside analog GS-5734 [417]. ZEBOV virions have been found in

the semen of male Ebola survivors, which can cause acute EVD if transmitted to their partner,

suggesting ZEBOV virus transmission can be similar to other sexually transmitted viruses [360].

Hence clinical trials testing the efficacy of nucleoside analogues to reduce the shedding of

virions in the semen of male Ebola survivors are currently underway: a phase II FPV trial in

Guinea and a phase II GS-5734 trial in Liberia [419].

194

The successful rVSV-ZEBOV-GP cluster-randomized ring vaccine trial in Guinea, which

protected 100% of those immeditaly vaccinated (N = 2,119) compared with those receiving

delayed vaccination (N = 2,041) [26], may provide protection in future ZEBOV outbreaks for

health care workers and quarantined contacts of EVD patients. Until the vaccine demonstrates

durable protection and broad neutralization of the five Ebolavirus species (ZEBOV, SUDV,

BDBV, RESTV and TAFV) a future outbreak emerging from a new EBOV strain may be

resistant to the current rVSV-ZEBOV-GP formulation. Thus, the rVSV-ZEBOV-GP vaccine is

one of many components in the EBOV toolkit needed to reduce the spread of future infections.

Government cooperation, effective border screening of EVD symptoms, robust healthcare

infrastructure, extensive training of healthcare personnel, proper Personel Protective Equipment

(PPE), rapid onsite EBOV antigen testing, contact tracing, community engagement, public health

education, and access to the basic necessities of life, all contribute to the epidemiological spread

and control of an EBOV epidemic. Thus, an effective treatment or combination of treatments

could have far reaching consequences in the transmission of EBOV in addition to immediate

patient care. An effective treatment could provide psychological reassurance that improves trust

between healthcare staff and ill patients, encouraging people to seek treatment at an ETC and

reduce EBOV spread among family caregivers at home. An emergent EBOV zoonose may also

show higher lethality compared to the ZEBOV-Makona and ZEBOV-Kikwit strains of the most

recent EBOV outbreak. For all of these reasons, future research into effective nucleoside analog

drug combinations should continue to proceed in preclinical in vivo models of EBOV infection,

until there is an effective treatment for acute and chronic EBOV infection.

195

References

196

1. de Souza MS, Ratto-Kim S, Chuenarom W, et al. The Thai phase III trial (RV144)

vaccine regimen induces T cell responses that preferentially target epitopes within the V2

region of HIV-1 envelope. J Immunol 2012; 188:5166-5176.

2. UNAIDS, World Health Organization. A framework for voluntary medical male

circumcision: Effective HIV prevention and a gateway to improved adolescent boys’ &

men’s health in eastern and southern Africa by 2021. 2016.

http://www.who.int/hiv/pub/malecircumcision/vmmc-policy-2016/en/.

3. Zhang L, Ramratnam B, Tenner-Racz K, et al. Quantifying residual HIV-1 replication in

patients receiving combination antiretroviral therapy. N Engl J Med 1999; 340:1605-

1613.

4. GARP, UNAIDS. Global AIDS Update. 2016.

http://www.unaids.org/en/resources/documents/2016/Global-AIDS-update-2016

5. World Health Organization. Ebola Situation Report, 10 June 2016. 2016.

http://apps.who.int/ebola/ebola-situation-reports

6. Madelain V, Nguyen TH, Olivo A, et al. Ebola Virus Infection: Review of the

Pharmacokinetic and Pharmacodynamic Properties of Drugs Considered for Testing in

Human Efficacy Trials. Clin Pharmacokinet 2016; 55:907-923.

7. Solomon DA, Sax PE. Current state and limitations of daily oral therapy for treatment.

Curr Opin HIV AIDS 2015; 10:219-225.

8. Public Health Agency of Canada. Summary: Estimates of HIV Incidence, Prevalence and

Proportion Undiagnosed in Canada, 2014. 2015.

http://www.catie.ca/sites/default/files/2014-HIV-Estimates-in-Canada-EN.pdf

9. Archin NM, Vaidya NK, Kuruc JD, et al. Immediate antiviral therapy appears to restrict

resting CD4+ cell HIV-1 infection without accelerating the decay of latent infection.

Proc Natl Acad Sci U S A 2012; 109:9523-9528.

10. Ma Q, Vaida F, Wong J, et al. Long-term efavirenz use is associated with worse

neurocognitive functioning in HIV-infected patients. J Neurovirol 2015; 22:170-178.

11. Gibb DM, Kizito H, Russell EC, et al. Pregnancy and infant outcomes among HIV-

infected women taking long-term ART with and without tenofovir in the DART trial.

PLoS Med 2012; 9:e1001217.

12. Scherzer R, Estrella M, Li Y, et al. Association of tenofovir exposure with kidney disease

risk in HIV infection. Aids 2012; 26:867-875.

13. El-Sherif O, Khoo S, Solas C. Key drug-drug interactions with direct-acting antiviral in

HIV-HCV coinfection. Curr Opin HIV AIDS 2015; 10:348-354.

197

14. von Moltke LL, Greenblatt DJ, Grassi JM, et al. Protease inhibitors as inhibitors of

human cytochromes P450: high risk associated with ritonavir. J Clin Pharmacol 1998;

38:106-111.

15. Marzolini C, Gibbons S, Khoo S, et al. Cobicistat versus ritonavir boosting and

differences in the drug-drug interaction profiles with co-medications. J Antimicrob

Chemother 2016; 71:1755-1758.

16. Leligdowicz A, Fischer WA, 2nd, Uyeki TM, et al. Ebola virus disease and critical

illness. Crit Care 2016; 20:10.1186.

17. Mendoza EJ, Qiu X, Kobinger GP. Progression of Ebola Therapeutics During the 2014-

2015 Outbreak. Trends Mol Med 2016; 22:164-173.

18. Sissoko D, Laouenan C, Folkesson E, et al. Experimental Treatment with Favipiravir for

Ebola Virus Disease (the JIKI Trial): A Historically Controlled, Single-Arm Proof-of-

Concept Trial in Guinea. PLoS Med 2016; 13:e1001967.

19. Davey RT, Jr., Dodd L, Proschan MA, et al. A Randomized, Controlled Trial of ZMapp

for Ebola Virus Infection. N Engl J Med 2016; 375:1448-1456.

20. van Griensven J, Edwards T, de Lamballerie X, et al. Evaluation of Convalescent Plasma

for Ebola Virus Disease in Guinea. N Engl J Med 2016; 374:33-42.

21. Thi EP, Mire CE, Lee AC, et al. Lipid nanoparticle siRNA treatment of Ebola-virus-

Makona-infected nonhuman primates. Nature 2015; 521:362-365.

22. Konde MK, Baker DP, Traore FA, et al. Interferon beta-1a for the treatment of Ebola

virus disease: A historically controlled, single-arm proof-of-concept trial. PLoS One

2017; 12:e0169255.

23. Oestereich L, Ludtke A, Wurr S, et al. Successful treatment of advanced Ebola virus

infection with T-705 (favipiravir) in a small animal model. Antiviral Res 2014; 105:17-

21.

24. Dunning J, Kennedy SB, Antierens A, et al. Experimental Treatment of Ebola Virus

Disease with Brincidofovir. PLoS One 2016; 11:e0162199.

25. Cardile AP, Downey LG, Wiseman PD, et al. Antiviral therapeutics for the treatment of

Ebola virus infection. Curr Opin Pharmacol 2016; 30:138-143.

26. Henao-Restrepo AM, Camacho A, Longini IM, et al. Efficacy and effectiveness of an

rVSV-vectored vaccine in preventing Ebola virus disease: final results from the Guinea

ring vaccination, open-label, cluster-randomised trial (Ebola Ca Suffit!). Lancet 2016;

389:505-518.

198

27. Dunning J, Sahr F, Rojek A, et al. Experimental Treatment of Ebola Virus Disease with

TKM-130803: A Single-Arm Phase 2 Clinical Trial. PLoS Med 2016; 13:e1001997.

28. McMullan LK, Flint M, Dyall J, et al. The lipid moiety of brincidofovir is required for in

vitro antiviral activity against Ebola virus. Antiviral Res 2016; 125:71-78.

29. Smither SJ, Eastaugh LS, Steward JA, et al. Post-exposure efficacy of oral T-705

(Favipiravir) against inhalational Ebola virus infection in a mouse model. Antiviral Res

2014; 104:153-155.

30. Qiu X, Wong G, Audet J, et al. Reversion of advanced Ebola virus disease in nonhuman

primates with ZMapp. Nature 2014; 514:47-53.

31. Smith LM, Hensley LE, Geisbert TW, et al. Interferon-beta therapy prolongs survival in

rhesus macaque models of Ebola and Marburg hemorrhagic fever. J Infect Dis 2013;

208:310-318.

32. Stanley DA, Honko AN, Asiedu C, et al. Chimpanzee adenovirus vaccine generates acute

and durable protective immunity against ebolavirus challenge. Nat Med 2014; 20:1126-

1129.

33. Qiu X, Fernando L, Alimonti JB, et al. Mucosal immunization of cynomolgus macaques

with the VSVDeltaG/ZEBOVGP vaccine stimulates strong ebola GP-specific immune

responses. PLoS One 2009; 4:e5547.

34. Curran JW. Pneumocystis pneumonia--Los Angeles. MMWR Morb Mortal Wkly Rep

1981; 30:250-252.

35. Quagliarello V. The Acquired Immunodeficiency Syndrome: current status. Yale J Biol

Med 1982; 55:443-452.

36. Barre-Sinoussi F, Chermann JC, Rey F, et al. Isolation of a T-lymphotropic retrovirus

from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 1983;

220:868-871.

37. Vilmer E, Barre-Sinoussi F, Rouzioux C, et al. Isolation of new lymphotropic retrovirus

from two siblings with haemophilia B, one with AIDS. Lancet 1984; 1:753-757.

38. Gallo RC, Salahuddin SZ, Popovic M, et al. Frequent detection and isolation of

cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS.

Science 1984; 224:500-503.

39. Sarngadharan MG, Popovic M, Bruch L, et al. Antibodies reactive with human T-

lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 1984;

224:506-508.

199

40. Keele BF, Van Heuverswyn F, Li Y, et al. Chimpanzee reservoirs of pandemic and

nonpandemic HIV-1. Science 2006; 313:523-526.

41. Jespersen S, Tolstrup M, Honge BL, et al. High level of HIV-1 drug resistance among

patients with HIV-1 and HIV-1/2 dual infections in Guinea-Bissau. J Virol 2015;

12:10.1186.

42. Vermund SH, Hayes RJ. Combination prevention: new hope for stopping the epidemic.

Curr HIV/AIDS Rep 2013; 10:169-186.

43. Faria NR, Rambaut A, Suchard MA, et al. HIV epidemiology. The early spread and

epidemic ignition of HIV-1 in human populations. Science 2014; 346:56-61.

44. Disease NIoAaI, National Institutes Health. How HIV Causes AIDS. 2004.

45. Suzuki Y, Suzuki Y. Gene Regulatable Lentiviral Vector System. In: Viral Gene

Therapy. Edited by Xu K. Rijeka: InTech; 2011. pp. 10.5772.

46. Brian Foley TL, Cristian Apetrei, Beatrice Hahn, Ilene Mizrachi, James Mullins, Andrew

Rambaut, Steven Wolinsky, and Bette Korber. HIV Sequence Compendium 2016. In. Los

Alamos, New Mexico.: Los Alamos National Laboratory; 2016.

47. Kwong PD, Wyatt R, Robinson J, et al. Structure of an HIV gp120 envelope glycoprotein

in complex with the CD4 receptor and a neutralizing human antibody. Nature 1998;

393:648-659.

48. Coakley E, Petropoulos CJ, Whitcomb JM. Assessing chemokine co-receptor usage in

HIV. Curr Opin Infect Dis 2005; 18:9-15.

49. Lawson VA, Oelrichs R, Guillon C, et al. Adaptive changes after human

immunodeficiency virus type 1 transmission. AIDS Res Hum Retroviruses 2002; 18:545-

556.

50. Miyauchi K, Kim Y, Latinovic O, et al. HIV enters cells via endocytosis and dynamin-

dependent fusion with endosomes. Cell 2009; 137:433-444.

51. Furuta RA, Wild CT, Weng Y, et al. Capture of an early fusion-active conformation of

HIV-1 gp41. Nat Struct Biol 1998; 5:276-279.

52. Spear M, Guo J, Turner A, et al. HIV-1 triggers WAVE2 phosphorylation in primary

CD4 T cells and macrophages, mediating Arp2/3-dependent nuclear migration. J Biol

Chem 2014; 289:6949-6959.

53. Coiras M, Lopez-Huertas MR, Perez-Olmeda M, et al. Understanding HIV-1 latency

provides clues for the eradication of long-term reservoirs. Nat Rev Microbiol 2009;

7:798-812.

200

54. Jayappa KD, Ao Z, Yao X. The HIV-1 passage from cytoplasm to nucleus: the process

involving a complex exchange between the components of HIV-1 and cellular machinery

to access nucleus and successful integration. Int J Biochem Mol Biol 2012; 3:70-85.

55. Daniel R, Katz RA, Skalka AM. A role for DNA-PK in retroviral DNA integration.

Science 1999; 284:644-647.

56. Bukrinsky M, Sharova N, Stevenson M. Human immunodeficiency virus type 1 2-LTR

circles reside in a nucleoprotein complex which is different from the preintegration

complex. J Virol 1993; 67:6863-6865.

57. Bukrinsky MI, Stanwick TL, Dempsey MP, et al. Quiescent T lymphocytes as an

inducible virus reservoir in HIV-1 infection. Science 1991; 254:423-427.

58. Sweet T, Sawaya BE, Khalili K, et al. Interplay between NFBP and NF-kappaB

modulates tat activation of the LTR. J Cell Physiol 2005; 204:375-380.

59. Jayaraman B, Crosby DC, Homer C, et al. RNA-directed remodeling of the HIV-1

protein Rev orchestrates assembly of the Rev-Rev response element complex. Elife 2014;

3:e04120.

60. Fenouillet E, Jones IM. The glycosylation of human immunodeficiency virus type 1

transmembrane glycoprotein (gp41) is important for the efficient intracellular transport of

the envelope precursor gp160. J Gen Virol 1995; 76:1509-1514.

61. Pfeiffer T, Zentgraf H, Freyaldenhoven B, et al. Transfer of endoplasmic reticulum and

Golgi retention signals to human immunodeficiency virus type 1 gp160 inhibits

intracellular transport and proteolytic processing of viral glycoprotein but does not

influence the cellular site of virus particle budding. J Gen Virol 1997; 78:1745-1753.

62. Borsetti A, Ohagen A, Gottlinger HG. The C-terminal half of the human

immunodeficiency virus type 1 Gag precursor is sufficient for efficient particle assembly.

J Virol 1998; 72:9313-9317.

63. Wen X, Ding L, Wang JJ, et al. ROCK1 and LIM kinase modulate retrovirus particle

release and cell-cell transmission events. J Virol 2014; 88:6906-6921.

64. Liang C, Hu J, Russell RS, et al. Translation of Pr55(gag) augments packaging of human

immunodeficiency virus type 1 RNA in a cis-acting manner. AIDS Res Hum Retroviruses

2002; 18:1117-1126.

65. Linde ME, Colquhoun DR, Ubaida Mohien C, et al. The conserved set of host proteins

incorporated into HIV-1 virions suggests a common egress pathway in multiple cell

types. J Proteome Res 2013; 12:2045-2054.

201

66. Tessmer U, Krausslich HG. Cleavage of human immunodeficiency virus type 1

proteinase from the N-terminally adjacent p6* protein is essential for efficient Gag

polyprotein processing and viral infectivity. J Virol 1998; 72:3459-3463.

67. Ayehunie S, Groves RW, Bruzzese AM, et al. Acutely infected Langerhans cells are

more efficient than T cells in disseminating HIV type 1 to activated T cells following a

short cell-cell contact. AIDS Res Hum Retroviruses 1995; 11:877-884.

68. Gurney KB, Elliott J, Nassanian H, et al. Binding and transfer of human

immunodeficiency virus by DC-SIGN+ cells in human rectal mucosa. J Virol 2005;

79:5762-5773.

69. Turville SG, Cameron PU, Handley A, et al. Diversity of receptors binding HIV on

dendritic cell subsets. Nat Immunol 2002; 3:975-983.

70. Kwon DS, Gregorio G, Bitton N, et al. DC-SIGN-mediated internalization of HIV is

required for trans-enhancement of T cell infection. Immunity 2002; 16:135-144.

71. Cameron PU, Freudenthal PS, Barker JM, et al. Dendritic cells exposed to human

immunodeficiency virus type-1 transmit a vigorous cytopathic infection to CD4+ T cells.

Science 1992; 257:383-387.

72. McDonald D, Wu L, Bohks SM, et al. Recruitment of HIV and its receptors to dendritic

cell-T cell junctions. Science 2003; 300:1295-1297.

73. Wiley RD, Gummuluru S. Immature dendritic cell-derived exosomes can mediate HIV-1

trans infection. Proc Natl Acad Sci U S A 2006; 103:738-743.

74. Nobile C, Petit C, Moris A, et al. Covert human immunodeficiency virus replication in

dendritic cells and in DC-SIGN-expressing cells promotes long-term transmission to

lymphocytes. J Virol 2005; 79:5386-5399.

75. Sharpless NE, O'Brien WA, Verdin E, et al. Human immunodeficiency virus type 1

tropism for brain microglial cells is determined by a region of the env glycoprotein that

also controls macrophage tropism. J Virol 1992; 66:2588-2593.

76. Alouf JE, Geoffroy C, Klatzmann D, et al. High production of the acquired

immunodeficiency syndrome virus (lymphadenopathy-associated virus) by human T

lymphocytes stimulated by streptococcal mitogenic toxins. J Clin Microbiol 1986;

24:639-641.

77. Gerstoft J, Petersen CS, Kroon S, et al. The immunological and clinical outcome of HIV

infection: 31 months of follow-up in a cohort of homosexual men. Scand J Infect Dis

1987; 19:503-509.

202

78. Jones NA, Wei X, Flower DR, et al. Determinants of human immunodeficiency virus

type 1 escape from the primary CD8+ cytotoxic T lymphocyte response. J Exp Med

2004; 200:1243-1256.

79. Wei X, Decker JM, Wang S, et al. Antibody neutralization and escape by HIV-1. Nature

2003; 422:307-312.

80. Kim CJ, McKinnon LR, Kovacs C, et al. Mucosal Th17 cell function is altered during

HIV infection and is an independent predictor of systemic immune activation. J Immunol

2013; 191:2164-2173.

81. Nazli A, Chan O, Dobson-Belaire WN, et al. Exposure to HIV-1 directly impairs

mucosal epithelial barrier integrity allowing microbial translocation. PLoS Pathog 2010;

6:e1000852.

82. Brenchley JM, Price DA, Schacker TW, et al. Microbial translocation is a cause of

systemic immune activation in chronic HIV infection. Nat Med 2006; 12:1365-1371.

83. Catalfamo M, Wilhelm C, Tcheung L, et al. CD4 and CD8 T cell immune activation

during chronic HIV infection: roles of homeostasis, HIV, type I IFN, and IL-7. J

Immunol 2011; 186:2106-2116.

84. Rothen M, Gratzl S, Hirsch HH, et al. Apoptosis in HIV-infected individuals is an early

marker occurring independently of high viremia. AIDS Res Hum Retroviruses 1997;

13:771-779.

85. Nardelli B, Gonzalez CJ, Schechter M, et al. CD4+ blood lymphocytes are rapidly killed

in vitro by contact with autologous human immunodeficiency virus-infected cells. Proc

Natl Acad Sci U S A 1995; 92:7312-7316.

86. Zeng M, Smith AJ, Wietgrefe SW, et al. Cumulative mechanisms of lymphoid tissue

fibrosis and T cell depletion in HIV-1 and SIV infections. J Clin Invest 2011; 121:998-

1008.

87. Day CL, Kaufmann DE, Kiepiela P, et al. PD-1 expression on HIV-specific T cells is

associated with T-cell exhaustion and disease progression. Nature 2006; 443:350-354.

88. Chomont N, El-Far M, Ancuta P, et al. HIV reservoir size and persistence are driven by T

cell survival and homeostatic proliferation. Nat Med 2009; 15:893-900.

89. Langford D, Marquie-Beck J, de Almeida S, et al. Relationship of antiretroviral treatment

to postmortem brain tissue viral load in human immunodeficiency virus-infected patients.

J Neurovirol 2006; 12:100-107.

203

90. Lamers SL, Rose R, Maidji E, et al. HIV DNA Is Frequently Present within Pathologic

Tissues Evaluated at Autopsy from Combined Antiretroviral Therapy-Treated Patients

with Undetectable Viral Loads. J Virol 2016; 90:8968-8983.

91. Chun TW, Engel D, Berrey MM, et al. Early establishment of a pool of latently infected,

resting CD4(+) T cells during primary HIV-1 infection. Proc Natl Acad Sci U S A 1998;

95:8869-8873.

92. Margolis DM, Garcia JV, Hazuda DJ, et al. Latency reversal and viral clearance to cure

HIV-1. Science 2016; 353:aaf6517.

93. Parks WP, Parks ES, Fischl MA, et al. HIV-1 inhibition by azidothymidine in a

concurrently randomized placebo-controlled trail. J Acquir Immune Defic Syndr 1988;

1:125-130.

94. Chaisson RE, Allain JP, Leuther M, et al. Significant changes in HIV antigen level in the

serum of patients treated with azidothymidine. N Engl J Med 1986; 315:1610-1611.

95. Fischl MA, Richman DD, Grieco MH, et al. The efficacy of azidothymidine (AZT) in the

treatment of patients with AIDS and AIDS-related complex. A double-blind, placebo-

controlled trial. N Engl J Med 1987; 317:185-191.

96. Fischl MA, Parker CB, Pettinelli C, et al. A randomized controlled trial of a reduced

daily dose of zidovudine in patients with the acquired immunodeficiency syndrome. The

AIDS Clinical Trials Group. N Engl J Med 1990; 323:1009-1014.

97. Reiss P, Lange JM, Boucher CA, et al. Resumption of HIV antigen production during

continuous zidovudine treatment. Lancet 1988; 1:421.

98. Roberts JD, Bebenek K, Kunkel TA. The accuracy of reverse transcriptase from HIV-1.

Science 1988; 242:1171-1173.

99. Larder BA, Kemp SD. Multiple mutations in HIV-1 reverse transcriptase confer high-

level resistance to zidovudine (AZT). Science 1989; 246:1155-1158.

100. Maxeiner HG, Keulen W, Schuurman R, et al. Selection of zidovudine resistance

mutations and escape of human immunodeficiency virus type 1 from antiretroviral

pressure in stavudine-treated pediatric patients. J Infect Dis 2002; 185:1070-1076.

101. La Seta Catamancio S, De Pasquale MP, Citterio P, et al. In vitro evolution of the human

immunodeficiency virus type 1 gag-protease region and maintenance of reverse

transcriptase resistance following prolonged drug exposure. J Clin Microbiol 2001;

39:1124-1129.

204

102. Eron JJ, Benoit SL, Jemsek J, et al. Treatment with lamivudine, zidovudine, or both in

HIV-positive patients with 200 to 500 CD4+ cells per cubic millimeter. North American

HIV Working Party. N Engl J Med 1995; 333:1662-1669.

103. D'Aquila RT, Hughes MD, Johnson VA, et al. Nevirapine, zidovudine, and didanosine

compared with zidovudine and didanosine in patients with HIV-1 infection. A

randomized, double-blind, placebo-controlled trial. National Institute of Allergy and

Infectious Diseases AIDS Clinical Trials Group Protocol 241 Investigators. Ann Intern

Med 1996; 124:1019-1030.

104. Collier AC, Coombs RW, Schoenfeld DA, et al. Combination therapy with zidovudine,

didanosine and saquinavir. Antiviral Res 1996; 29:99.

105. Granich R, Gupta S, Hersh B, et al. Trends in AIDS Deaths, New Infections and ART

Coverage in the Top 30 Countries with the Highest AIDS Mortality Burden; 1990-2013.

PLoS One 2015; 10:e0131353.

106. World Health Organization. Consolidated guidelines on the use of antiretroviral drugs for

treating and preventing HIV infection: Recommendations for a public health approach.

2016. http://www.who.int/hiv/pub/arv/arv-2016/en/

107. Kearney MF, Wiegand A, Shao W, et al. Origin of Rebound Plasma HIV Includes Cells

with Identical Proviruses That Are Transcriptionally Active before Stopping of

Antiretroviral Therapy. J Virol 2015; 90:1369-1376.

108. Gray GE, Laher F, Lazarus E, et al. Approaches to preventative and therapeutic HIV

vaccines. Curr Opin Virol 2016; 17:104-109.

109. Kaminski R, Bella R, Yin C, et al. Excision of HIV-1 DNA by gene editing: a proof-of-

concept in vivo study. Gene Ther 2016; 23:690-695.

110. Wang CX, Cannon PM. Clinical Applications of Genome Editing to HIV Cure. AIDS

Patient Care STDS 2016; 30:539-544.

111. Flynn NM, Forthal DN, Harro CD, et al. Placebo-controlled phase 3 trial of a

recombinant glycoprotein 120 vaccine to prevent HIV-1 infection. J Infect Dis 2005;

191:654-665.

112. Spearman P, Lally MA, Elizaga M, et al. A trimeric, V2-deleted HIV-1 envelope

glycoprotein vaccine elicits potent neutralizing antibodies but limited breadth of

neutralization in human volunteers. J Infect Dis 2011; 203:1165-1173.

113. De Rosa SC, Thomas EP, Bui J, et al. HIV-DNA priming alters T cell responses to HIV-

adenovirus vaccine even when responses to DNA are undetectable. J Immunol 2011;

187:3391-3401.

205

114. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, et al. Vaccination with ALVAC and

AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 2009; 361:2209-2220.

115. Caskey M, Klein F, Lorenzi JC, et al. Viraemia suppressed in HIV-1-infected humans by

broadly neutralizing antibody 3BNC117. Nature 2015; 522:487-491.

116. Moodie Z, Metch B, Bekker LG, et al. Continued Follow-Up of Phambili Phase 2b

Randomized HIV-1 Vaccine Trial Participants Supports Increased HIV-1 Acquisition

among Vaccinated Men. PLoS One 2015; 10:e0137666.

117. Lazarus EM, Otwombe K, Adonis T, et al. Uptake of genital mucosal sampling in HVTN

097, a phase 1b HIV vaccine trial in South Africa. PLoS One 2014; 9:e112303.

118. Chun TW, Murray D, Justement JS, et al. Broadly neutralizing antibodies suppress HIV

in the persistent viral reservoir. Proc Natl Acad Sci U S A 2014; 111:13151-13156.

119. Alexandre KB, Mufhandu HT, London GM, et al. Progress and Perspectives on HIV-1

microbicide development. Virology 2016; 497:69-80.

120. Riddler SA, Husnik M, Gorbach PM, et al. Long-term follow-up of HIV seroconverters

in microbicide trials - rationale, study design, and challenges in MTN-015. HIV Clin

Trials 2016; 17:204-211.

121. Montgomery CM, Gafos M, Lees S, et al. Re-framing microbicide acceptability: findings

from the MDP301 trial. Cult Health Sex 2010; 12:649-662.

122. Greene E, Batona G, Hallad J, et al. Acceptability and adherence of a candidate

microbicide gel among high-risk women in Africa and India. Cult Health Sex 2010;

12:739-754.

123. Martin M, Vanichseni S, Suntharasamai P, et al. The impact of adherence to preexposure

prophylaxis on the risk of HIV infection among people who inject drugs. AIDS 2015;

29:819-824.

124. Denton PW, Othieno F, Martinez-Torres F, et al. One percent tenofovir applied topically

to humanized BLT mice and used according to the CAPRISA 004 experimental design

demonstrates partial protection from vaginal HIV infection, validating the BLT model for

evaluation of new microbicide candidates. J Virol 2011; 85:7582-7593.

125. Molina JM, Capitant C, Spire B, et al. On-Demand Preexposure Prophylaxis in Men at

High Risk for HIV-1 Infection. N Engl J Med 2015; 373:2237-2246.

126. Kaminski R, Chen Y, Fischer T, et al. Elimination of HIV-1 Genomes from Human T-

lymphoid Cells by CRISPR/Cas9 Gene Editing. Sci Rep 2016; 6:10.1038.

206

127. Wang Z, Pan Q, Gendron P, et al. CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1

Replication and Accelerate Viral Escape. Cell Rep 2016; 15:481-489.

128. Tsunetsugu-Yokota Y, Kobayahi-Ishihara M, Wada Y, et al. Homeostatically Maintained

Resting Naive CD4+ T Cells Resist Latent HIV Reactivation. Front Microbiol 2016;

7:10.3389.

129. Deng K, Pertea M, Rongvaux A, et al. Broad CTL response is required to clear latent

HIV-1 due to dominance of escape mutations. Nature 2015; 517:381-385.

130. Laird GM, Bullen CK, Rosenbloom DI, et al. Ex vivo analysis identifies effective HIV-1

latency-reversing drug combinations. J Clin Invest 2015; 125:1901-1912.

131. Weydert C, Rijck JD, Christ F, et al. Targeting Virus-host Interactions of HIV

Replication. Curr Top Med Chem 2016; 16:1167-1190.

132. Cooper DA, Heera J, Ive P, et al. Efficacy and safety of maraviroc vs. efavirenz in

treatment-naive patients with HIV-1: 5-year findings. AIDS 2014; 28:717-725.

133. Reynes J, Arasteh K, Clotet B, et al. TORO: ninety-six-week virologic and immunologic

response and safety evaluation of enfuvirtide with an optimized background of

antiretrovirals. AIDS Patient Care STDS 2007; 21:533-543.

134. Dorr P, Westby M, Dobbs S, et al. Maraviroc (UK-427,857), a potent, orally

bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with

broad-spectrum anti-human immunodeficiency virus type 1 activity. Antimicrob Agents

Chemother 2005; 49:4721-4732.

135. Readinger JA, Schiralli GM, Jiang JK, et al. Selective targeting of ITK blocks multiple

steps of HIV replication. Proc Natl Acad Sci U S A 2008; 105:6684-6689.

136. Wrasidlo W, Crews LA, Tsigelny IF, et al. Neuroprotective effects of the anti-cancer

drug sunitinib in models of HIV neurotoxicity suggests potential for the treatment of

neurodegenerative disorders. Br J Pharmacol 2014; 171:5757-5773.

137. van Dijk D, Ertaylan G, Boucher CA, et al. Identifying potential survival strategies of

HIV-1 through virus-host protein interaction networks. BMC Syst Biol 2010; 4:10.1186.

138. Jager S, Cimermancic P, Gulbahce N, et al. Global landscape of HIV-human protein

complexes. Nature 2012; 481:365-370.

139. Smyth RP, Negroni M. A step forward understanding HIV-1 diversity. Retrovirology

2016; 13:27.

207

140. Sugden SM, Bego MG, Pham TN, et al. Remodeling of the Host Cell Plasma Membrane

by HIV-1 Nef and Vpu: A Strategy to Ensure Viral Fitness and Persistence. Viruses

2016; 8.

141. Simon V, Bloch N, Landau NR. Intrinsic host restrictions to HIV-1 and mechanisms of

viral escape. Nat Immunol 2015; 16:546-553.

142. Mangeat B, Turelli P, Caron G, et al. Broad antiretroviral defence by human

APOBEC3G through lethal editing of nascent reverse transcripts. Nature 2003; 424:99-

103.

143. Goldstone DC, Ennis-Adeniran V, Hedden JJ, et al. HIV-1 restriction factor SAMHD1 is

a deoxynucleoside triphosphate triphosphohydrolase. Nature 2011; 480:379-382.

144. Vranckx LS, Demeulemeester J, Saleh S, et al. LEDGIN-mediated Inhibition of

Integrase-LEDGF/p75 Interaction Reduces Reactivation of Residual Latent HIV.

EBioMedicine 2016; 8:248-264.

145. Christ F, Voet A, Marchand A, et al. Rational design of small-molecule inhibitors of the

LEDGF/p75-integrase interaction and HIV replication. Nat Chem Biol 2010; 6:442-448.

146. Bertoletti F, Crespan E, Maga G. Tyrosine kinases as essential cellular cofactors and

potential therapeutic targets for human immunodeficiency virus infection. Cell Mol Biol

(Noisy-le-grand) 2012; 58:31-43.

147. Davis CB, Dikic I, Unutmaz D, et al. Signal transduction due to HIV-1 envelope

interactions with chemokine receptors CXCR4 or CCR5. J Exp Med 1997; 186:1793-

1798.

148. Cayota A, Vuillier F, Siciliano J, et al. Defective protein tyrosine phosphorylation and

altered levels of p59fyn and p56lck in CD4 T cells from HIV-1 infected patients. Int

Immunol 1994; 6:611-621.

149. Phipps DJ, Yousefi S, Branch DR. Increased enzymatic activity of the T-cell antigen

receptor-associated fyn protein tyrosine kinase in asymptomatic patients infected with the

human immunodeficiency virus. Blood 1997; 90:3603-3612.

150. Guo J, Xu X, Rasheed TK, et al. Genistein interferes with SDF-1- and HIV-mediated

actin dynamics and inhibits HIV infection of resting CD4 T cells. Retrovirology 2013;

10:10.1186.

151. Stantchev TS, Markovic I, Telford WG, et al. The tyrosine kinase inhibitor genistein

blocks HIV-1 infection in primary human macrophages. Virus Res 2007; 123:178-189.

208

152. Schiralli Lester GM, Akiyama H, Evans E, et al. Interleukin 2-inducible T cell kinase

(ITK) facilitates efficient egress of HIV-1 by coordinating Gag distribution and actin

organization. Virology 2012.

153. Zack JA, Arrigo SJ, Weitsman SR, et al. HIV-1 entry into quiescent primary

lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 1990; 61:213-

222.

154. Bukong TN, Hall WW, Jacque JM. Lentivirus-associated MAPK/ERK2 phosphorylates

EMD and regulates infectivity. J Gen Virol 2010; 91:2381-2392.

155. Francis AC, Di Primio C, Allouch A, et al. Role of phosphorylation in the nuclear

biology of HIV-1. Curr Med Chem 2011; 18:2904-2912.

156. Pauls E, Badia R, Torres-Torronteras J, et al. Palbociclib, a selective inhibitor of cyclin-

dependent kinase4/6, blocks HIV-1 reverse transcription through the control of sterile

alpha motif and HD domain-containing protein-1 (SAMHD1) activity. AIDS 2014;

28:2213-2222.

157. Dochi T, Nakano T, Inoue M, et al. Phosphorylation of human immunodeficiency virus

type 1 capsid protein at serine 16, required for peptidyl-prolyl isomerase-dependent

uncoating, is mediated by virion-incorporated extracellular signal-regulated kinase 2. J

Gen Virol 2014; 95:1156-1166.

158. Gallay P, Swingler S, Aiken C, et al. HIV-1 infection of nondividing cells: C-terminal

tyrosine phosphorylation of the viral matrix protein is a key regulator. Cell 1995; 80:379-

388.

159. Manganaro L, Lusic M, Gutierrez MI, et al. Concerted action of cellular JNK and Pin1

restricts HIV-1 genome integration to activated CD4+ T lymphocytes. Nat Med 2010;

16:329-333.

160. Lau A, Swinbank KM, Ahmed PS, et al. Suppression of HIV-1 infection by a small

molecule inhibitor of the ATM kinase. Nat Cell Biol 2005; 7:493-500.

161. Flory E, Weber CK, Chen P, et al. Plasma membrane-targeted Raf kinase activates NF-

kappaB and human immunodeficiency virus type 1 replication in T lymphocytes. J Virol

1998; 72:2788-2794.

162. Meggio F, D'Agostino DM, Ciminale V, et al. Phosphorylation of HIV-1 Rev protein:

implication of protein kinase CK2 and pro-directed kinases. Biochem Biophys Res

Commun 1996; 226:547-554.

163. Schubert U, Strebel K. Differential activities of the human immunodeficiency virus type

1-encoded Vpu protein are regulated by phosphorylation and occur in different cellular

compartments. J Virol 1994; 68:2260-2271.

209

164. Trible RP, Emert-Sedlak L, Smithgall TE. HIV-1 Nef selectively activates Src family

kinases Hck, Lyn, and c-Src through direct SH3 domain interaction. J Biol Chem 2006;

281:27029-27038.

165. Chang AH, O'Shaughnessy MV, Jirik FR. Hck SH3 domain-dependent abrogation of

Nef-induced class 1 MHC down-regulation. Eur J Immunol 2001; 31:2382-2387.

166. Hung CH, Thomas L, Ruby CE, et al. HIV-1 Nef assembles a Src family kinase-ZAP-

70/Syk-PI3K cascade to downregulate cell-surface MHC-I. Cell Host Microbe 2007;

1:121-133.

167. Pan X, Geist MM, Rudolph JM, et al. HIV-1 Nef disrupts membrane-microdomain-

associated anterograde transport for plasma membrane delivery of selected Src family

kinases. Cell Microbiol 2013; 15:1605-1621.

168. Hanna Z, Weng X, Kay DG, et al. The pathogenicity of human immunodeficiency virus

(HIV) type 1 Nef in CD4C/HIV transgenic mice is abolished by mutation of its SH3-

binding domain, and disease development is delayed in the absence of Hck. J Virol 2001;

75:9378-9392.

169. Wu P, Nielsen TE, Clausen MH. Small-molecule kinase inhibitors: an analysis of FDA-

approved drugs. Drug Discov Today 2016; 21:5-10.

170. Wu P, Nielsen TE, Clausen MH. FDA-approved small-molecule kinase inhibitors. Trends

Pharmacol Sci 2015; 36:422-439.

171. Richeldi L, du Bois RM, Raghu G, et al. Efficacy and safety of nintedanib in idiopathic

pulmonary fibrosis. N Engl J Med 2014; 370:2071-2082.

172. Asahina A, Etoh T, Igarashi A, et al. Oral tofacitinib efficacy, safety and tolerability in

Japanese patients with moderate to severe plaque psoriasis and psoriatic arthritis: A

randomized, double-blind, phase 3 study. J Dermatol 2016.

173. Spivak AM, Larragoite ET, Coletti ML, et al. Janus kinase inhibition suppresses PKC-

induced cytokine release without affecting HIV-1 latency reversal ex vivo. Retrovirology

2016; 13:10.1186.

174. Worm SW, Bower M, Reiss P, et al. Non-AIDS defining cancers in the D:A:D Study--

time trends and predictors of survival: a cohort study. BMC Infect Dis 2013; 13:471.

175. Kowalkowski MA, Day RS, Du XL, et al. Cumulative HIV viremia and non-AIDS-

defining malignancies among a sample of HIV-infected male veterans. J Acquir Immune

Defic Syndr 2014; 67:204-211.

176. Koon HB, Bubley GJ, Pantanowitz L, et al. Imatinib-induced regression of AIDS-related

Kaposi's sarcoma. J Clin Oncol 2005; 23:982-989.

210

177. Hagiwara S, Yotsumoto M, Odawara T, et al. Non-AIDS-defining hematological

malignancies in HIV-infected patients: an epidemiological study in Japan. Aids 2013;

27:279-283.

178. de Wispelaere M, LaCroix AJ, Yang PL. The small molecules AZD0530 and dasatinib

inhibit dengue virus RNA replication via Fyn kinase. J Virol 2013; 87:7367-7381.

179. Guntermann C, Zheng R, Nye KE. The effects of CD3, CD4 and CD28 signaling on

lymphocytes during human immunodeficiency virus-1 infection. Eur J Immunol 1997;

27:1966-1972.

180. Maqueda A, Moyano JV, Gutierrez-Lopez MD, et al. Activation pathways of

alpha4beta1 integrin leading to distinct T-cell cytoskeleton reorganization, Rac1

regulation and Pyk2 phosphorylation. J Cell Physiol 2006; 207:746-756.

181. Sol-Foulon N, Sourisseau M, Porrot F, et al. ZAP-70 kinase regulates HIV cell-to-cell

spread and virological synapse formation. Embo j 2007; 26:516-526.

182. Liu J, Liao Z, Camden J, et al. Src homology 3 binding sites in the P2Y2 nucleotide

receptor interact with Src and regulate activities of Src, proline-rich tyrosine kinase 2,

and growth factor receptors. J Biol Chem 2004; 279:8212-8218.

183. Wu JC, Chen YC, Kuo CT, et al. Focal adhesion kinase-dependent focal adhesion

recruitment of SH2 domains directs SRC into focal adhesions to regulate cell adhesion

and migration. Sci Rep 2015; 5:10.1038.

184. Bain J, McLauchlan H, Elliott M, et al. The specificities of protein kinase inhibitors: an

update. Biochem J 2003; 371:199-204.

185. Rous P. A SARCOMA OF THE FOWL TRANSMISSIBLE BY AN AGENT

SEPARABLE FROM THE TUMOR CELLS. J Exp Med 1911; 13:397-411.

186. Stehelin D, Varmus HE, Bishop JM, et al. DNA related to the transforming gene(s) of

avian sarcoma viruses is present in normal avian DNA. Nature 1976; 260:170-173.

187. Pawson T, Kung TH, Martin GS. Structure and phosphorylation of the Fujinami sarcoma

virus gene product. J Virol 1981; 40:665-672.

188. DeClue JE, Sadowski I, Martin GS, et al. A conserved domain regulates interactions of

the v-fps protein-tyrosine kinase with the host cell. Proc Natl Acad Sci U S A 1987;

84:9064-9068.

189. Bjorge JD, Jakymiw A, Fujita DJ. Selected glimpses into the activation and function of

Src kinase. Oncogene 2000; 19:5620-5635.

211

190. Cayer MP, Proulx M, Ma XZ, et al. c-Src tyrosine kinase co-associates with and

phosphorylates signal transducer and activator of transcription 5b which mediates the

proliferation of normal human B lymphocytes. Clin Exp Immunol 2009; 156:419-427.

191. Kaplan KB, Bibbins KB, Swedlow JR, et al. Association of the amino-terminal half of c-

Src with focal adhesions alters their properties and is regulated by phosphorylation of

tyrosine 527. Embo j 1994; 13:4745-4756.

192. Bergman M, Mustelin T, Oetken C, et al. The human p50csk tyrosine kinase

phosphorylates p56lck at Tyr-505 and down regulates its catalytic activity. Embo j 1992;

11:2919-2924.

193. Kedzierska K, Vardaxis NJ, Jaworowski A, et al. FcgammaR-mediated phagocytosis by

human macrophages involves Hck, Syk, and Pyk2 and is augmented by GM-CSF. J

Leukoc Biol 2001; 70:322-328.

194. Takata M, Sabe H, Hata A, et al. Tyrosine kinases Lyn and Syk regulate B cell receptor-

coupled Ca2+ mobilization through distinct pathways. Embo j 1994; 13:1341-1349.

195. Tronick SR, Popescu NC, Cheah MS, et al. Isolation and chromosomal localization of the

human fgr protooncogene, a distinct member of the tyrosine kinase gene family. Proc

Natl Acad Sci U S A 1985; 82:6595-6599.

196. Compeer EB, Janssen W, van Royen-Kerkhof A, et al. Dysfunctional BLK in common

variable immunodeficiency perturbs B-cell proliferation and ability to elicit antigen-

specific CD4+ T-cell help. Oncotarget 2015; 6:10759-10771.

197. Dikic I, Tokiwa G, Lev S, et al. A role for Pyk2 and Src in linking G-protein-coupled

receptors with MAP kinase activation. Nature 1996; 383:547-550.

198. Okada M. Regulation of the SRC family kinases by Csk. Int J Biol Sci 2012; 8:1385-

1397.

199. Engen JR, Wales TE, Hochrein JM, et al. Structure and dynamic regulation of Src-family

kinases. Cell Mol Life Sci 2008; 65:3058-3073.

200. Nakashima K, Takeuchi K, Chihara K, et al. HCV NS5A protein containing potential

ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src

family kinase Fyn in B cells. PLoS One 2012; 7:e46634.

201. Hirsch AJ, Medigeshi GR, Meyers HL, et al. The Src family kinase c-Yes is required for

maturation of West Nile virus particles. J Virol 2005; 79:11943-11951.

202. Pfannkuche A, Buther K, Karthe J, et al. c-Src is required for complex formation between

the hepatitis C virus-encoded proteins NS5A and NS5B: a prerequisite for replication.

Hepatology 2011; 53:1127-1136.

212

203. Delorme-Axford E, Sadovsky Y, Coyne CB. Lipid raft- and SRC family kinase-

dependent entry of coxsackievirus B into human placental trophoblasts. J Virol 2013;

87:8569-8581.

204. Majolini MB, D'Elios MM, Galieni P, et al. Expression of the T-cell-specific tyrosine

kinase Lck in normal B-1 cells and in chronic lymphocytic leukemia B cells. Blood 1998;

91:3390-3396.

205. Moroi Y, Koga Y, Nakamura K, et al. Induction of interleukin 2-responsiveness in

thymocytes of the transgenic mice carrying lck-transgene. Microbiol Immunol 1993;

37:369-381.

206. Einspahr KJ, Abraham RT, Dick CJ, et al. Protein tyrosine phosphorylation and p56lck

modification in IL-2 or phorbol ester-activated human natural killer cells. J Immunol

1990; 145:1490-1497.

207. Omri B, Crisanti P, Marty MC, et al. The Lck tyrosine kinase is expressed in brain

neurons. J Neurochem 1996; 67:1360-1364.

208. Ley SC, Marsh M, Bebbington CR, et al. Distinct intracellular localization of Lck and

Fyn protein tyrosine kinases in human T lymphocytes. J Cell Biol 1994; 125:639-649.

209. Yousefi S, Ma XZ, Singla R, et al. HIV-1 infection is facilitated in T cells by decreasing

p56lck protein tyrosine kinase activity. Clin Exp Immunol 2003; 133:78-90.

210. Barber EK, Dasgupta JD, Schlossman SF, et al. The CD4 and CD8 antigens are coupled

to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex. Proc Natl

Acad Sci U S A 1989; 86:3277-3281.

211. Veillette A, Bookman MA, Horak EM, et al. The CD4 and CD8 T cell surface antigens

are associated with the internal membrane tyrosine-protein kinase p56lck. Cell 1988;

55:301-308.

212. Luo KX, Sefton BM. Cross-linking of T-cell surface molecules CD4 and CD8 stimulates

phosphorylation of the lck tyrosine protein kinase at the autophosphorylation site. Mol

Cell Biol 1990; 10:5305-5313.

213. Straus DB, Weiss A. Genetic evidence for the involvement of the lck tyrosine kinase in

signal transduction through the T cell antigen receptor. Cell 1992; 70:585-593.

214. Phipps DJ, Read SE, Piovesan JP, et al. HIV infection in vitro enhances the activity of

src-family protein tyrosine kinases. AIDS 1996; 10:1191-1198.

215. Briant L, Robert-Hebmann V, Acquaviva C, et al. The protein tyrosine kinase p56lck is

required for triggering NF-kappaB activation upon interaction of human

213

immunodeficiency virus type 1 envelope glycoprotein gp120 with cell surface CD4. J

Virol 1998; 72:6207-6214.

216. Hivroz C, Mazerolles F, Soula M, et al. Human immunodeficiency virus gp120 and

derived peptides activate protein tyrosine kinase p56lck in human CD4 T lymphocytes.

Eur J Immunol 1993; 23:600-607.

217. Juszczak RJ, Turchin H, Truneh A, et al. Effect of human immunodeficiency virus gp120

glycoprotein on the association of the protein tyrosine kinase p56lck with CD4 in human

T lymphocytes. J Biol Chem 1991; 266:11176-11183.

218. Trushin SA, Bren GD, Badley AD. CXCR4 Tropic HIV-1 gp120 Inhibition of SDF-

1alpha-Induced Chemotaxis Requires Lck and is Associated with Cofilin

Phosphorylation. Open Virol J 2010; 4:157-162.

219. Mazerolles F, Barbat C, Fischer A. Down-regulation of LFA-1-mediated T cell adhesion

induced by the HIV envelope glycoprotein gp160 requires phosphatidylinositol-3-kinase

activity. Eur J Immunol 1997; 27:2457-2465.

220. Chrobak P, Simard MC, Bouchard N, et al. HIV-1 Nef disrupts maturation of CD4+ T

cells through CD4/Lck modulation. J Immunol 2010; 185:3948-3959.

221. Fackler OT, Alcover A, Schwartz O. Modulation of the immunological synapse: a key to

HIV-1 pathogenesis? Nat Rev Immunol 2007; 7:310-317.

222. Laguette N, Bregnard C, Bouchet J, et al. Nef-induced CD4 endocytosis in human

immunodeficiency virus type 1 host cells: role of p56lck kinase. J Virol 2009; 83:7117-

7128.

223. Pan X, Rudolph JM, Abraham L, et al. HIV-1 Nef compensates for disorganization of the

immunological synapse by inducing trans-Golgi network-associated Lck signaling. Blood

2012; 119:786-797.

224. Haller C, Muller B, Fritz JV, et al. HIV-1 Nef and Vpu are functionally redundant broad-

spectrum modulators of cell surface receptors, including tetraspanins. J Virol 2014;

88:14241-14257.

225. Wolf D, Witte V, Clark P, et al. HIV Nef enhances Tat-mediated viral transcription

through a hnRNP-K-nucleated signaling complex. Cell Host Microbe 2008; 4:398-408.

226. Manna SK, Aggarwal BB. Differential requirement for p56lck in HIV-tat versus TNF-

induced cellular responses: effects on NF-kappa B, activator protein-1, c-Jun N-terminal

kinase, and apoptosis. J Immunol 2000; 164:5156-5166.

227. Strasner AB, Natarajan M, Doman T, et al. The Src kinase Lck facilitates assembly of

HIV-1 at the plasma membrane. J Immunol 2008; 181:3706-3713.

214

228. Len AC, Starling S, Shivkumar M, et al. HIV-1 Activates T Cell Signaling Independently

of Antigen to Drive Viral Spread. Cell Rep 2017; 18:1062-1074.

229. Samelson LE, Phillips AF, Luong ET, et al. Association of the fyn protein-tyrosine

kinase with the T-cell antigen receptor. Proc Natl Acad Sci U S A 1990; 87:4358-4362.

230. Xie YG, Yu Y, Hou LK, et al. FYN promotes breast cancer progression through

epithelial-mesenchymal transition. Oncol Rep 2016; 36:1000-1006.

231. Peckham H, Giuffrida L, Wood R, et al. Fyn is an intermediate kinase that BDNF utilizes

to promote oligodendrocyte myelination. Glia 2016; 64:255-269.

232. Davidson D, Viallet J, Veillette A. Unique catalytic properties dictate the enhanced

function of p59fynT, the hemopoietic cell-specific isoform of the Fyn tyrosine protein

kinase, in T cells. Mol Cell Biol 1994; 14:4554-4564.

233. Hunter AJ, Shimizu Y. Alpha 4 beta 1 integrin-mediated tyrosine phosphorylation in

human T cells: characterization of Crk- and Fyn-associated substrates (pp105, pp115, and

human enhancer of filamentation-1) and integrin-dependent activation of p59fyn1. J

Immunol 1997; 159:4806-4814.

234. da Silva AJ, Li Z, de Vera C, et al. Cloning of a novel T-cell protein FYB that binds FYN

and SH2-domain-containing leukocyte protein 76 and modulates interleukin 2

production. Proc Natl Acad Sci U S A 1997; 94:7493-7498.

235. Sharma N, Akhade AS, Qadri A. Src kinases central to T-cell receptor signaling regulate

TLR-activated innate immune responses from human T cells. Innate Immun 2016;

22:238-244.

236. Tamma SM, Chirmule N, McCloskey TW, et al. Signals transduced through the CD4

molecule interfere with TCR/CD3-mediated ras activation leading to T cell

anergy/apoptosis. Clin Immunol Immunopathol 1997; 85:195-201.

237. Hohashi N, Hayashi T, Fusaki N, et al. The protein tyrosine kinase Fyn activates

transcription from the HIV promoter via activation of NF kappa B-like DNA-binding

proteins. Int Immunol 1995; 7:1851-1859.

238. Douaisi M, Dussart S, Courcoul M, et al. The tyrosine kinases Fyn and Hck favor the

recruitment of tyrosine-phosphorylated APOBEC3G into vif-defective HIV-1 particles.

Biochem Biophys Res Commun 2005; 329:917-924.

239. Quintrell N, Lebo R, Varmus H, et al. Identification of a human gene (HCK) that encodes

a protein-tyrosine kinase and is expressed in hemopoietic cells. Mol Cell Biol 1987;

7:2267-2275.

215

240. Dale BM, Traum D, Erdjument-Bromage H, et al. Phagocytosis in macrophages lacking

Cbl reveals an unsuspected role for Fc gamma receptor signaling and actin assembly in

target binding. J Immunol 2009; 182:5654-5662.

241. Dwyer AR, Mouchemore KA, Steer JH, et al. Src family kinase expression and

subcellular localization in macrophages: implications for their role in CSF-1-induced

macrophage migration. J Leukoc Biol 2016; 100:163-175.

242. Smolinska MJ, Page TH, Urbaniak AM, et al. Hck tyrosine kinase regulates TLR4-

induced TNF and IL-6 production via AP-1. J Immunol 2011; 187:6043-6051.

243. Mohn H, Le Cabec V, Fischer S, et al. The src-family protein-tyrosine kinase p59hck is

located on the secretory granules in human neutrophils and translocates towards the

phagosome during cell activation. Biochem J 1995; 309:657-665.

244. Jelicic K, Cimbro R, Nawaz F, et al. The HIV-1 envelope protein gp120 impairs B cell

proliferation by inducing TGF-beta1 production and FcRL4 expression. Nat Immunol

2013; 14:1256-1265.

245. Komuro I, Yokota Y, Yasuda S, et al. CSF-induced and HIV-1-mediated distinct

regulation of Hck and C/EBPbeta represent a heterogeneous susceptibility of monocyte-

derived macrophages to M-tropic HIV-1 infection. J Exp Med 2003; 198:443-453.

246. Tintori C, Laurenzana I, La Rocca F, et al. Identification of Hck inhibitors as hits for the

development of antileukemia and anti-HIV agents. ChemMedChem 2013; 8:1353-1360.

247. Breuer S, Espinola S, Morelli X, et al. A Biochemical/Biophysical Assay Dyad for HTS-

Compatible Triaging of Inhibitors of the HIV-1 Nef/Hck SH3 Interaction. Curr Chem

Genom Transl Med 2013; 7:16-20.

248. Hiyoshi M, Suzu S, Yoshidomi Y, et al. Interaction between Hck and HIV-1 Nef

negatively regulates cell surface expression of M-CSF receptor. Blood 2008; 111:243-

250.

249. Hiyoshi M, Takahashi-Makise N, Yoshidomi Y, et al. HIV-1 Nef perturbs the function,

structure, and signaling of the Golgi through the Src kinase Hck. J Cell Physiol 2012;

227:1090-1097.

250. Shinya E, Shimizu M, Owaki A, et al. Hemopoietic cell kinase (Hck) and p21-activated

kinase 2 (PAK2) are involved in the down-regulation of CD1a lipid antigen presentation

by HIV-1 Nef in dendritic cells. Virology 2016; 487:285-295.

251. Cornall A, Mak J, Greenway A, et al. HIV-1 infection of T cells and macrophages are

differentially modulated by virion-associated Hck: a Nef-dependent phenomenon.

Viruses 2013; 5:2235-2252.

216

252. Verollet C, Souriant S, Bonnaud E, et al. HIV-1 reprograms the migration of

macrophages. Blood 2015; 125:1611-1622.

253. Cassol E, Rossouw T, Malfeld S, et al. CD14(+) macrophages that accumulate in the

colon of African AIDS patients express pro-inflammatory cytokines and are responsive to

lipopolysaccharide. BMC Infect Dis 2015; 15:10.1186.

254. Batisse J, Guerrero SX, Bernacchi S, et al. APOBEC3G impairs the multimerization of

the HIV-1 Vif protein in living cells. J Virol 2013; 87:6492-6506.

255. Hassaine G, Courcoul M, Bessou G, et al. The tyrosine kinase Hck is an inhibitor of

HIV-1 replication counteracted by the viral vif protein. J Biol Chem 2001; 276:16885-

16893.

256. Yamanashi Y, Mori S, Yoshida M, et al. Selective expression of a protein-tyrosine

kinase, p56lyn, in hematopoietic cells and association with production of human T-cell

lymphotropic virus type I. Proc Natl Acad Sci U S A 1989; 86:6538-6542.

257. Balasubramanian A, Ganju RK, Groopman JE. Signal transducer and activator of

transcription factor 1 mediates apoptosis induced by hepatitis C virus and HIV envelope

proteins in hepatocytes. J Infect Dis 2006; 194:670-681.

258. Hernandez-Rapp J, Martin-Lanneree S, Hirsch TZ, et al. A PrP(C)-caveolin-Lyn complex

negatively controls neuronal GSK3beta and serotonin 1B receptor. Sci Rep 2014;

4:10.1038.

259. Kawakami Y, Kitaura J, Satterthwaite AB, et al. Redundant and opposing functions of

two tyrosine kinases, Btk and Lyn, in mast cell activation. J Immunol 2000; 165:1210-

1219.

260. Yamanashi Y, Kakiuchi T, Mizuguchi J, et al. Association of B cell antigen receptor with

protein tyrosine kinase Lyn. Science 1991; 251:192-194.

261. Nishizumi H, Taniuchi I, Yamanashi Y, et al. Impaired proliferation of peripheral B cells

and indication of autoimmune disease in lyn-deficient mice. Immunity 1995; 3:549-560.

262. Rolli V, Gallwitz M, Wossning T, et al. Amplification of B cell antigen receptor

signaling by a Syk/ITAM positive feedback loop. Mol Cell 2002; 10:1057-1069.

263. Narute PS, Smithgall TE. Nef alleles from all major HIV-1 clades activate Src-family

kinases and enhance HIV-1 replication in an inhibitor-sensitive manner. PLoS One 2012;

7:e32561.

264. Gilbert C, Barat C, Cantin R, et al. Involvement of Src and Syk tyrosine kinases in HIV-1

transfer from dendritic cells to CD4+ T lymphocytes. J Immunol 2007; 178:2862-2871.

217

265. Caparros E, Munoz P, Sierra-Filardi E, et al. DC-SIGN ligation on dendritic cells results

in ERK and PI3K activation and modulates cytokine production. Blood 2006; 107:3950-

3958.

266. Ellegard R, Crisci E, Burgener A, et al. Complement opsonization of HIV-1 results in

decreased antiviral and inflammatory responses in immature dendritic cells via CR3. J

Immunol 2014; 193:4590-4601.

267. Tomkowicz B, Lee C, Ravyn V, et al. The Src kinase Lyn is required for CCR5 signaling

in response to MIP-1beta and R5 HIV-1 gp120 in human macrophages. Blood 2006;

108:1145-1150.

268. Cheung R, Ravyn V, Wang L, et al. Signaling mechanism of HIV-1 gp120 and virion-

induced IL-1beta release in primary human macrophages. J Immunol 2008; 180:6675-

6684.

269. Malik M, Chen YY, Kienzle MF, et al. Monocyte migration and LFA-1-mediated

attachment to brain microvascular endothelia is regulated by SDF-1 alpha through Lyn

kinase. J Immunol 2008; 181:4632-4637.

270. Berton G, Fumagalli L, Laudanna C, et al. Beta 2 integrin-dependent protein tyrosine

phosphorylation and activation of the FGR protein tyrosine kinase in human neutrophils.

J Cell Biol 1994; 126:1111-1121.

271. Kim HS, Han HD, Armaiz-Pena GN, et al. Functional roles of Src and Fgr in ovarian

carcinoma. Clin Cancer Res 2011; 17:1713-1721.

272. Willman CL, Stewart CC, Longacre TL, et al. Expression of the c-fgr and hck protein-

tyrosine kinases in acute myeloid leukemic blasts is associated with early commitment

and differentiation events in the monocytic and granulocytic lineages. Blood 1991;

77:726-734.

273. King FJ, Cole MD. Molecular cloning and sequencing of the murine c-fgr gene.

Oncogene 1990; 5:337-344.

274. Fanibunda SE, Modi DN, Bandivdekar AH. HIV gp120 induced gene expression

signatures in vaginal epithelial cells. Microbes Infect 2013; 15:806-815.

275. Sugawara K, Sugawara I, Sukegawa J, et al. Distribution of c-yes-1 gene product in

various cells and tissues. Br J Cancer 1991; 63:508-513.

276. Semba K, Yamanashi Y, Nishizawa M, et al. Location of the c-yes gene on the human

chromosome and its expression in various tissues. Science 1985; 227:1038-1040.

218

277. Fuhrer DK, Yang YC. Complex formation of JAK2 with PP2A, P13K, and Yes in

response to the hematopoietic cytokine interleukin-11. Biochem Biophys Res Commun

1996; 224:289-296.

278. Yoder SM, Dineen SL, Wang Z, et al. YES, a Src family kinase, is a proximal glucose-

specific activator of cell division cycle control protein 42 (Cdc42) in pancreatic islet beta

cells. J Biol Chem 2014; 289:11476-11487.

279. Iida M, Brand TM, Campbell DA, et al. Yes and Lyn play a role in nuclear translocation

of the epidermal growth factor receptor. Oncogene 2013; 32:759-767.

280. Siegbahn A, Johnell M, Nordin A, et al. TF/FVIIa transactivate PDGFRbeta to regulate

PDGF-BB-induced chemotaxis in different cell types: involvement of Src and PLC.

Arterioscler Thromb Vasc Biol 2008; 28:135-141.

281. Varrin-Doyer M, Vincent P, Cavagna S, et al. Phosphorylation of collapsin response

mediator protein 2 on Tyr-479 regulates CXCL12-induced T lymphocyte migration. J

Biol Chem 2009; 284:13265-13276.

282. Johnson TP, Patel K, Johnson KR, et al. Induction of IL-17 and nonclassical T-cell

activation by HIV-Tat protein. Proc Natl Acad Sci U S A 2013; 110:13588-13593.

283. Wang X, Viswanath R, Zhao J, et al. Changes in the level of apoptosis-related proteins in

Jurkat cells infected with HIV-1 versus HIV-2. Mol Cell Biochem 2010; 337:175-183.

284. Dymecki SM, Niederhuber JE, Desiderio SV. Specific expression of a tyrosine kinase

gene, blk, in B lymphoid cells. Science 1990; 247:332-336.

285. Borowiec M, Liew CW, Thompson R, et al. Mutations at the BLK locus linked to

maturity onset diabetes of the young and beta-cell dysfunction. Proc Natl Acad Sci U S A

2009; 106:14460-14465.

286. Saijo K, Schmedt C, Su IH, et al. Essential role of Src-family protein tyrosine kinases in

NF-kappaB activation during B cell development. Nat Immunol 2003; 4:274-279.

287. Tretter T, Ross AE, Dordai DI, et al. Mimicry of pre-B cell receptor signaling by

activation of the tyrosine kinase Blk. J Exp Med 2003; 198:1863-1873.

288. Simpfendorfer KR, Armstead BE, Shih A, et al. Autoimmune disease-associated

haplotypes of BLK exhibit lowered thresholds for B cell activation and expansion of Ig

class-switched B cells. Arthritis Rheumatol 2015; 67:2866-2876.

289. Konig R, Zhou Y, Elleder D, et al. Global analysis of host-pathogen interactions that

regulate early-stage HIV-1 replication. Cell 2008; 135:49-60.

219

290. Pyper JM, Bolen JB. Neuron-specific splicing of C-SRC RNA in human brain. J

Neurosci Res 1989; 24:89-96.

291. Golden A, Nemeth SP, Brugge JS. Blood platelets express high levels of the pp60c-src-

specific tyrosine kinase activity. Proc Natl Acad Sci U S A 1986; 83:852-856.

292. Nishio H, Tokuda M, Itano T, et al. pp60c-src expression in rat spermatogenesis.

Biochem Biophys Res Commun 1995; 206:502-510.

293. Branch DR, Mills GB. pp60c-src expression is induced by activation of normal human T

lymphocytes. J Immunol 1995; 154:3678-3685.

294. Amanchy R, Zhong J, Molina H, et al. Identification of c-Src tyrosine kinase substrates

using mass spectrometry and peptide microarrays. J Proteome Res 2008; 7:3900-3910.

295. Smart JE, Oppermann H, Czernilofsky AP, et al. Characterization of sites for tyrosine

phosphorylation in the transforming protein of Rous sarcoma virus (pp60v-src) and its

normal cellular homologue (pp60c-src). Proc Natl Acad Sci U S A 1981; 78:6013-6017.

296. Yang CC, Fazli L, Loguercio S, et al. Downregulation of c-SRC kinase CSK promotes

castration resistant prostate cancer and pinpoints a novel disease subclass. Oncotarget

2015; 6:22060-22071.

297. Sandilands E, Cans C, Fincham VJ, et al. RhoB and actin polymerization coordinate Src

activation with endosome-mediated delivery to the membrane. Dev Cell 2004; 7:855-869.

298. David-Pfeuty T, Nouvian-Dooghe Y. Highly specific antibody to Rous sarcoma virus src

gene product recognizes nuclear and nucleolar antigens in human cells. J Virol 1995;

69:1699-1713.

299. Weber TK, Steele G, Summerhayes IC. Differential pp60c-src activity in well and poorly

differentiated human colon carcinomas and cell lines. J Clin Invest 1992; 90:815-821.

300. Qian XL, Zhang J, Li PZ, et al. Dasatinib inhibits c-src phosphorylation and prevents the

proliferation of Triple-Negative Breast Cancer (TNBC) cells which overexpress

Syndecan-Binding Protein (SDCBP). PLoS One 2017; 12:e0171169.

301. Roussel RR, Brodeur SR, Shalloway D, et al. Selective binding of activated pp60c-src by

an immobilized synthetic phosphopeptide modeled on the carboxyl terminus of pp60c-

src. Proc Natl Acad Sci U S A 1991; 88:10696-10700.

302. Piwnica-Worms H, Saunders KB, Roberts TM, et al. Tyrosine phosphorylation regulates

the biochemical and biological properties of pp60c-src. Cell 1987; 49:75-82.

303. Ren R, Mayer BJ, Cicchetti P, et al. Identification of a ten-amino acid proline-rich SH3

binding site. Science 1993; 259:1157-1161.

220

304. Anderson D, Koch CA, Grey L, et al. Binding of SH2 domains of phospholipase C

gamma 1, GAP, and Src to activated growth factor receptors. Science 1990; 250:979-982.

305. Alexandropoulos K, Baltimore D. Coordinate activation of c-Src by SH3- and SH2-

binding sites on a novel p130Cas-related protein, Sin. Genes Dev 1996; 10:1341-1355.

306. Somani AK, Bignon JS, Mills GB, et al. Src kinase activity is regulated by the SHP-1

protein-tyrosine phosphatase. J Biol Chem 1997; 272:21113-21119.

307. Cooper JA, MacAuley A. Potential positive and negative autoregulation of p60c-src by

intermolecular autophosphorylation. Proc Natl Acad Sci U S A 1988; 85:4232-4236.

308. Zheng XM, Wang Y, Pallen CJ. Cell transformation and activation of pp60c-src by

overexpression of a protein tyrosine phosphatase. Nature 1992; 359:336-339.

309. Chellaiah MA, Schaller MD. Activation of Src kinase by protein-tyrosine phosphatase-

PEST in osteoclasts: comparative analysis of the effects of bisphosphonate and protein-

tyrosine phosphatase inhibitor on Src activation in vitro. J Cell Physiol 2009; 220:382-

393.

310. Oh ES, Gu H, Saxton TM, et al. Regulation of early events in integrin signaling by

protein tyrosine phosphatase SHP-2. Mol Cell Biol 1999; 19:3205-3215.

311. Chong YP, Mulhern TD, Cheng HC. C-terminal Src kinase (CSK) and CSK-homologous

kinase (CHK)--endogenous negative regulators of Src-family protein kinases. Growth

Factors 2005; 23:233-244.

312. Davidson D, Bakinowski M, Thomas ML, et al. Phosphorylation-dependent regulation of

T-cell activation by PAG/Cbp, a lipid raft-associated transmembrane adaptor. Mol Cell

Biol 2003; 23:2017-2028.

313. Tsukita S, Oishi K, Akiyama T, et al. Specific proto-oncogenic tyrosine kinases of src

family are enriched in cell-to-cell adherens junctions where the level of tyrosine

phosphorylation is elevated. J Cell Biol 1991; 113:867-879.

314. Fincham VJ, Unlu M, Brunton VG, et al. Translocation of Src kinase to the cell periphery

is mediated by the actin cytoskeleton under the control of the Rho family of small G

proteins. J Cell Biol 1996; 135:1551-1564.

315. Bard F, Patel U, Levy JB, et al. Molecular complexes that contain both c-Cbl and c-Src

associate with Golgi membranes. Eur J Cell Biol 2002; 81:26-35.

316. Finan PM, Hall A, Kellie S. Sam68 from an immortalised B-cell line associates with a

subset of SH3 domains. FEBS Lett 1996; 389:141-144.

221

317. He JJ, Henao-Mejia J, Liu Y. Sam68 functions in nuclear export and translation of HIV-1

RNA. RNA Biol 2009; 6:384-386.

318. Phipps DJ, Reed-Doob P, MacFadden DK, et al. An octapeptide analogue of HIV gp120

modulates protein tyrosine kinase activity in activated peripheral blood T lymphocytes.

Clin Exp Immunol 1995; 100:412-418.

319. Briggs SD, Lerner EC, Smithgall TE. Affinity of Src family kinase SH3 domains for HIV

Nef in vitro does not predict kinase activation by Nef in vivo. Biochemistry 2000;

39:489-495.

320. He JC, Husain M, Sunamoto M, et al. Nef stimulates proliferation of glomerular

podocytes through activation of Src-dependent Stat3 and MAPK1,2 pathways. J Clin

Invest 2004; 114:643-651.

321. Zhang X, Yu J, Kuzontkoski PM, et al. Slit2/Robo4 Signaling Modulates HIV-1 gp120-

Induced Lymphatic Hyperpermeability. PLoS Pathogens 2012; 8:e1002461.

322. Schlaepfer DD, Hauck CR, Sieg DJ. Signaling through focal adhesion kinase. Prog

Biophys Mol Biol 1999; 71:435-478.

323. Prasad A, Kuzontkoski PM, Shrivastava A, et al. Slit2N/Robo1 Inhibit HIV-gp120-

Induced Migration and Podosome Formation in Immature Dendritic Cells by

Sequestering LSP1 and WASp. PLoS One 2012; 7:e48854.

324. Xiong WC, Macklem M, Parsons JT. Expression and characterization of splice variants

of PYK2, a focal adhesion kinase-related protein. J Cell Sci 1998; 111:1981-1991.

325. Dikic I, Dikic I, Schlessinger J. Identification of a new Pyk2 isoform implicated in

chemokine and antigen receptor signaling. J Biol Chem 1998; 273:14301-14308.

326. Block ER, Tolino MA, Klarlund JK. Pyk2 activation triggers epidermal growth factor

receptor signaling and cell motility after wounding sheets of epithelial cells. J Biol Chem

2010; 285:13372-13379.

327. Sasaki H, Nagura K, Ishino M, et al. Cloning and characterization of cell adhesion kinase

beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily. J Biol Chem

1995; 270:21206-21219.

328. Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: in command and control

of cell motility. Nat Rev Mol Cell Biol 2005; 6:56-68.

329. Walkiewicz KW, Girault JA, Arold ST. How to awaken your nanomachines: Site-specific

activation of focal adhesion kinases through ligand interactions. Prog Biophys Mol Biol

2015; 119:60-71.

222

330. Avraham S, London R, Fu Y, et al. Identification and characterization of a novel related

adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain. J Biol Chem

1995; 270:27742-27751.

331. Vanarotti MS, Finkelstein DB, Guibao CD, et al. Structural basis for the interaction

between Pyk2-FAT domain and Leupaxin LD repeats. Biochemistry 2016; 55:1332-1345.

332. Riggs D, Yang Z, Kloss J, et al. The Pyk2 FERM regulates Pyk2 complex formation and

phosphorylation. Cell Signal 2011; 23:288-296.

333. Han S, Mistry A, Chang JS, et al. Structural characterization of proline-rich tyrosine

kinase 2 (PYK2) reveals a unique (DFG-out) conformation and enables inhibitor design.

J Biol Chem 2009; 284:13193-13201.

334. Litvak V, Tian D, Shaul YD, et al. Targeting of PYK2 to focal adhesions as a cellular

mechanism for convergence between integrins and G protein-coupled receptor signaling

cascades. J Biol Chem 2000; 275:32736-32746.

335. Melendez J, Turner C, Avraham H, et al. Cardiomyocyte apoptosis triggered by

RAFTK/pyk2 via Src kinase is antagonized by paxillin. J Biol Chem 2004; 279:53516-

53523.

336. Marzia M, Chiusaroli R, Neff L, et al. Calpain is required for normal osteoclast function

and is down-regulated by calcitonin. J Biol Chem 2006; 281:9745-9754.

337. Miao L, Xin X, Xin H, et al. Hydrogen Sulfide Recruits Macrophage Migration by

Integrin β1-Src-FAK/Pyk2-Rac Pathway in Myocardial Infarction. Sci Rep 2016;

6:10.1038.

338. Genua M, Xu S-Q, Buraschi S, et al. Proline-Rich Tyrosine Kinase 2 (Pyk2) Regulates

IGF-I-Induced Cell Motility and Invasion of Urothelial Carcinoma Cells. PLoS One

2012; 7:e40148.

339. Corvol JC, Valjent E, Toutant M, et al. Depolarization activates ERK and proline-rich

tyrosine kinase 2 (PYK2) independently in different cellular compartments in

hippocampal slices. J Biol Chem 2005; 280:660-668.

340. Collins M, Bartelt RR, Houtman JC. T cell receptor activation leads to two distinct

phases of Pyk2 activation and actin cytoskeletal rearrangement in human T cells. Mol

Immunol 2010; 47:1665-1674.

341. Beinke S, Phee H, Clingan JM, et al. Proline-rich tyrosine kinase-2 is critical for CD8 T-

cell short-lived effector fate. Proc Natl Acad Sci U S A 2010; 107:16234-16239.

342. Hashimoto-Tane A, Sakuma M, Ike H. Micro-adhesion rings surrounding TCR

microclusters are essential for T cell activation. J Exp Med 2016; 213:1609-1625.

223

343. Sancho D, Montoya MC, Monjas A, et al. TCR engagement induces proline-rich tyrosine

kinase-2 (Pyk2) translocation to the T cell-APC interface independently of Pyk2 activity

and in an immunoreceptor tyrosine-based activation motif-mediated fashion. J Immunol

2002; 169:292-300.

344. Tsuchida M, Knechtle SJ, Hamawy MM. CD28 ligation induces tyrosine phosphorylation

of Pyk2 but not Fak in Jurkat T cells. J Biol Chem 1999; 274:6735-6740.

345. Seror C, Melki MT, Subra F, et al. Extracellular ATP acts on P2Y2 purinergic receptors

to facilitate HIV-1 infection. J Exp Med 2011; 208:1823-1834.

346. Garron ML, Arthos J, Guichou JF, et al. Structural basis for the interaction between focal

adhesion kinase and CD4. J Mol Biol 2008; 375:1320-1328.

347. Cicala C, Arthos J, Ruiz M, et al. Induction of phosphorylation and intracellular

association of CC chemokine receptor 5 and focal adhesion kinase in primary human

CD4+ T cells by macrophage-tropic HIV envelope. J Immunol 1999; 163:420-426.

348. Cicala C, Arthos J, Rubbert A, et al. HIV-1 envelope induces activation of caspase-3 and

cleavage of focal adhesion kinase in primary human CD4(+) T cells. Proc Natl Acad Sci

U S A 2000; 97:1178-1183.

349. Preusser A, Briese L, Willbold D. Presence of a helix in human CD4 cytoplasmic domain

promotes binding to HIV-1 Nef protein. Biochem Biophys Res Commun 2002; 292:734-

740.

350. Simpson DIH. Ebola haemorrhagic fever in Sudan, 1976. Report of a WHO/International

Study Team. Bull World Health Organ 1978; 56:247-270.

351. Johnson KM, Breman JG. Ebola haemorrhagic fever in Zaire, 1976. Bull World Health

Organ 1978; 56:271-293.

352. Johnson KM, Lange JV, Webb PA, et al. Isolation and partial characterisation of a new

virus causing acute haemorrhagic fever in Zaire. Lancet 1977; 1:569-571.

353. Richman DD, Cleveland PH, McCormick JB, et al. Antigenic analysis of strains of Ebola

virus: identification of two Ebola virus serotypes. J Infect Dis 1983; 147:268-271.

354. Towner JS, Sealy TK, Khristova ML, et al. Newly discovered ebola virus associated with

hemorrhagic fever outbreak in Uganda. PLoS Pathog 2008; 4:e1000212.

355. Hayes CG, Burans JP, Ksiazek TG, et al. Outbreak of fatal illness among captive

macaques in the Philippines caused by an Ebola-related filovirus. Am J Trop Med Hyg

1992; 46:664-671.

224

356. Formenty P, Boesch C, Wyers M, et al. Ebola virus outbreak among wild chimpanzees

living in a rain forest of Cote d'Ivoire. J Infect Dis 1999; 179:S120-S126.

357. Rivera A, Messaoudi I. Molecular mechanisms of Ebola pathogenesis. J Leukoc Biol

2016; 100:889-904.

358. Baize S, Pannetier D, Oestereich L, et al. Emergence of Zaire Ebola virus disease in

Guinea. N Engl J Med 2014; 371:1418-1425.

359. Kuhn JH, Andersen KG, Baize S, et al. Nomenclature- and database-compatible names

for the two Ebola virus variants that emerged in Guinea and the Democratic Republic of

the Congo in 2014. Viruses 2014; 6:4760-4799.

360. Diallo B, Sissoko D, Loman NJ, et al. Resurgence of Ebola Virus Disease in Guinea

Linked to a Survivor With Virus Persistence in Seminal Fluid for More Than 500 Days.

Clin Infect Dis 2016; 63:1353-1356.

361. Leroy EM, Baize S, Volchkov VE, et al. Human asymptomatic Ebola infection and

strong inflammatory response. Lancet 2000; 355:2210-2215.

362. Tiffany A, Vetter P, Mattia J, et al. Ebola Virus Disease Complications as Experienced

by Survivors in Sierra Leone. Clin Infect Dis 2016; 62:1360-1366.

363. Hereth He'bert E, Oury Bah M, E'Tard J F, et al. Ocular complications in survivors of the

Ebola outbreak in Guinea. Am J Ophthalmol 2016; 175:114-121.

364. Clark DV, Kibuuka H, Millard M, et al. Long-term sequelae after Ebola virus disease in

Bundibugyo, Uganda: a retrospective cohort study. Lancet Infect Dis 2015; 15:905-912.

365. Sissoko D, Duraffour S, Kerber R, et al. Persistence and clearance of Ebola virus RNA

from seminal fluid of Ebola virus disease survivors: a longitudinal analysis and modelling

study. Lancet Glob Health 2017; 5:e80-e88.

366. Caluwaerts S, Fautsch T, Lagrou D, et al. Dilemmas in Managing Pregnant Women With

Ebola: 2 Case Reports. Clin Infect Dis 2016; 62:903-905.

367. Vetter P, Fischer WA, 2nd, Schibler M, et al. Ebola Virus Shedding and Transmission:

Review of Current Evidence. J Infect Dis 2016; 214:S177-S184.

368. Artimo P, Jonnalagedda M, Arnold K, et al. ExPASy: SIB bioinformatics resource portal.

Nucleic Acids Res 2012; 40:W597-603.

369. Sanchez A, Kiley MP, Holloway BP, et al. Sequence analysis of the Ebola virus genome:

organization, genetic elements, and comparison with the genome of Marburg virus. Virus

Res 1993; 29:215-240.

225

370. Neumann G, Watanabe S, Kawaoka Y. Characterization of Ebolavirus regulatory

genomic regions. Virus Res 2009; 144:1-7.

371. Sanchez A, Yang ZY, Xu L, et al. Biochemical analysis of the secreted and virion

glycoproteins of Ebola virus. J Virol 1998; 72:6442-6447.

372. Dahlmann F, Biedenkopf N, Babler A, et al. Analysis of Ebola Virus Entry Into

Macrophages. J Infect Dis 2015; 212:S247-257.

373. Takada A, Fujioka K, Tsuiji M, et al. Human macrophage C-type lectin specific for

galactose and N-acetylgalactosamine promotes filovirus entry. J Virol 2004; 78:2943-

2947.

374. Chan SY, Empig CJ, Welte FJ, et al. Folate receptor-alpha is a cofactor for cellular entry

by Marburg and Ebola viruses. Cell 2001; 106:117-126.

375. Aleksandrowicz P, Marzi A, Biedenkopf N, et al. Ebola virus enters host cells by

macropinocytosis and clathrin-mediated endocytosis. J Infect Dis 2011; 204:S957-967.

376. Aman MJ. Chasing Ebola through the Endosomal Labyrinth. MBio 2016; 7:e00346.

377. Wang H, Shi Y, Song J, et al. Ebola Viral Glycoprotein Bound to Its Endosomal

Receptor Niemann-Pick C1. Cell 2016; 164:258-268.

378. Nanbo A, Watanabe S, Halfmann P, et al. The spatio-temporal distribution dynamics of

Ebola virus proteins and RNA in infected cells. Sci Rep 2013; 3:10.1038.

379. Weik M, Modrof J, Klenk HD, et al. Ebola virus VP30-mediated transcription is

regulated by RNA secondary structure formation. J Virol 2002; 76:8532-8539.

380. Zinzula L, Esposito F, Pala D, et al. dsRNA binding characterization of full length

recombinant wild type and mutants Zaire ebolavirus VP35. Antiviral Res 2012; 93:354-

363.

381. Biedenkopf N, Lier C, Becker S. Dynamic Phosphorylation of VP30 Is Essential for

Ebola Virus Life Cycle. J Virol 2016; 90:4914-4925.

382. Licata JM, Johnson RF, Han Z, et al. Contribution of ebola virus glycoprotein,

nucleoprotein, and VP24 to budding of VP40 virus-like particles. J Virol 2004; 78:7344-

7351.

383. Yen BC, Basler CF. Effects of Filovirus Interferon Antagonists on Responses of Human

Monocyte-Derived Dendritic Cells to RNA Virus Infection. J Virol 2016; 90:5108-5118.

226

384. Hensley LE, Young HA, Jahrling PB, et al. Proinflammatory response during Ebola virus

infection of primate models: possible involvement of the tumor necrosis factor receptor

superfamily. Immunol Lett 2002; 80:169-179.

385. Stroher U, West E, Bugany H, et al. Infection and activation of monocytes by Marburg

and Ebola viruses. J Virol 2001; 75:11025-11033.

386. Gupta M, Mahanty S, Ahmed R, et al. Monocyte-derived human macrophages and

peripheral blood mononuclear cells infected with ebola virus secrete MIP-1alpha and

TNF-alpha and inhibit poly-IC-induced IFN-alpha in vitro. Virology 2001; 284:20-25.

387. Feldmann H, Geisbert TW. Ebola haemorrhagic fever. Lancet 2011; 377:849-862.

388. Lubaki NM, Ilinykh P, Pietzsch C, et al. The lack of maturation of Ebola virus-infected

dendritic cells results from the cooperative effect of at least two viral domains. J Virol

2013; 87:7471-7485.

389. Basler CF. A novel mechanism of immune evasion mediated by Ebola virus soluble

glycoprotein. Expert Rev Anti Infect Ther 2013; 11:475-478.

390. Geisbert TW, Hensley LE, Larsen T, et al. Pathogenesis of Ebola hemorrhagic fever in

cynomolgus macaques: evidence that dendritic cells are early and sustained targets of

infection. Am J Pathol 2003; 163:2347-2370.

391. Gupta M, Spiropoulou C, Rollin PE. Ebola virus infection of human PBMCs causes

massive death of macrophages, CD4 and CD8 T cell sub-populations in vitro. Virology

2007; 364:45-54.

392. Friedrich BM, Trefry JC, Biggins JE, et al. Potential vaccines and post-exposure

treatments for filovirus infections. Viruses 2012; 4:1619-1650.

393. Tapia MD, Sow SO, Lyke KE, et al. Use of ChAd3-EBO-Z Ebola virus vaccine in

Malian and US adults, and boosting of Malian adults with MVA-BN-Filo: a phase 1,

single-blind, randomised trial, a phase 1b, open-label and double-blind, dose-escalation

trial, and a nested, randomised, double-blind, placebo-controlled trial. Lancet Infect Dis

2016; 16:31-42.

394. Ewer K, Rampling T, Venkatraman N, et al. A Monovalent Chimpanzee Adenovirus

Ebola Vaccine Boosted with MVA. N Engl J Med 2016; 374:1635-1646.

395. Jones SM, Feldmann H, Stroher U, et al. Live attenuated recombinant vaccine protects

nonhuman primates against Ebola and Marburg viruses. Nat Med 2005; 11:786-790.

396. Jones SM, Stroher U, Fernando L, et al. Assessment of a vesicular stomatitis virus-based

vaccine by use of the mouse model of Ebola virus hemorrhagic fever. J Infect Dis 2007;

196:S404-412.

227

397. Feldmann H, Jones SM, Daddario-DiCaprio KM, et al. Effective post-exposure treatment

of Ebola infection. PLoS Pathog 2007; 3:e2.

398. Agnandji ST, Huttner A, Zinser ME, et al. Phase 1 Trials of rVSV Ebola Vaccine in

Africa and Europe. N Engl J Med 2016; 374:1647-1660.

399. Regules JA, Beigel JH, Paolino KM, et al. A Recombinant Vesicular Stomatitis Virus

Ebola Vaccine. N Engl J Med 2015; 376:330-331.

400. Gunther S, Feldmann H, Geisbert TW, et al. Management of accidental exposure to

Ebola virus in the biosafety level 4 laboratory, Hamburg, Germany. J Infect Dis 2011;

204:S785-790.

401. Lai L, Davey R, Beck A, et al. Emergency postexposure vaccination with vesicular

stomatitis virus-vectored Ebola vaccine after needlestick. JAMA 2015; 313:1249-1255.

402. van Griensven J, Edwards T, Baize S. Efficacy of Convalescent Plasma in Relation to

Dose of Ebola Virus Antibodies. N Engl J Med 2016; 375:2307-2309.

403. Geisbert TW, Lee AC, Robbins M, et al. Postexposure protection of non-human primates

against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study.

Lancet 2010; 375:1896-1905.

404. McCarthy M. US signs contract with ZMapp maker to accelerate development of the

Ebola drug. BMJ 2014; 349:g5488.

405. Basler CF, Amarasinghe GK. Evasion of interferon responses by Ebola and Marburg

viruses. J Interferon Cytokine Res 2009; 29:511-520.

406. Wong G, Kobinger GP, Qiu X. Characterization of host immune responses in Ebola virus

infections. Expert Rev Clin Immunol 2014; 10:781-790.

407. Lin FC, Young HA. Interferons: Success in anti-viral immunotherapy. Cytokine Growth

Factor Rev 2014; 25:369-376.

408. Eron JJ, Jr. The treatment of antiretroviral-naive subjects with the 3TC/zidovudine

combination: a review of North American (NUCA 3001) and European (NUCB 3001)

trials. AIDS 1996; 10:S11-19.

409. Cooper DA, Katlama C, Montaner J, et al. Randomised trial of addition of lamivudine or

lamivudine plus loviride to zidovudine-containing regimens for patients with HIV-1

infection: the CAESAR trial. Lancet 1997; 349:1413-1421.

410. Thio CL, Smeaton L, Hollabaugh K, et al. Comparison of HBV-active HAART regimens

in an HIV-HBV multinational cohort: outcomes through 144 weeks. AIDS 2015;

29:1173-1182.

228

411. Strand A, Bottiger D, Gever LN, et al. Safety and tolerability of combination acyclovir

5% and hydrocortisone 1% cream in adolescents with recurrent herpes simplex labialis.

Pediatr Dermatol 2012; 29:105-110.

412. Madkan VK, Arora A, Babb-Tarbox M, et al. Open-label study of valacyclovir 1.5 g

twice daily for the treatment of uncomplicated herpes zoster in immunocompetent

patients 18 years of age or older. J Cutan Med Surg 2007; 11:89-98.

413. Marty FM, Winston DJ, Rowley SD, et al. CMX001 to prevent cytomegalovirus disease

in hematopoietic-cell transplantation. N Engl J Med 2013; 369:1227-1236.

414. Wedemeyer H, Forns X, Hezode C, et al. Mericitabine and Either Boceprevir or

Telaprevir in Combination with Peginterferon Alfa-2a plus Ribavirin for Patients with

Chronic Hepatitis C Genotype 1 Infection and Prior Null Response: The Randomized

DYNAMO 1 and DYNAMO 2 Studies. PLoS One 2016; 11:e0145409.

415. Else LJ, Jackson A, Puls R, et al. Pharmacokinetics of lamivudine and lamivudine-

triphosphate after administration of 300 milligrams and 150 milligrams once daily to

healthy volunteers: results of the ENCORE 2 study. Antimicrob Agents Chemother 2012;

56:1427-1433.

416. Parang K, Wiebe LI, Knaus EE. Pharmacokinetics and tissue distribution of (+/-)-3'-

azido-2',3'-dideoxy-5'-O-(2-bromomyristoyl)thymidine, a prodrug of 3'-azido-2',3'-

dideoxythymidine (AZT) in mice. J Pharm Pharmacol 1998; 50:989-996.

417. Warren TK, Jordan R, Lo MK, et al. Therapeutic efficacy of the small molecule GS-5734

against Ebola virus in rhesus monkeys. Nature 2016; 531:381-385.

418. Warren TK, Wells J, Panchal RG, et al. Protection against filovirus diseases by a novel

broad-spectrum nucleoside analogue BCX4430. Nature 2014; 508:402-405.

419. Siegel D, Hui HC, Doerffler E, et al. Discovery and Synthesis of a Phosphoramidate

Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the

Treatment of Ebola and Emerging Viruses. J Med Chem 2017; 60:1648-1661.

420. Parker S, Touchette E, Oberle C, et al. Efficacy of therapeutic intervention with an oral

ether-lipid analogue of cidofovir (CMX001) in a lethal mousepox model. Antiviral Res

2008; 77:39-49.

421. Bainbridge JW, Raina J, Shah SM, et al. Ocular complications of intravenous cidofovir

for cytomegalovirus retinitis in patients with AIDS. Eye 1999; 13:353-356.

422. Kern ER, Hartline C, Harden E, et al. Enhanced inhibition of orthopoxvirus replication in

vitro by alkoxyalkyl esters of cidofovir and cyclic cidofovir. Antimicrob Agents

Chemother 2002; 46:991-995.

229

423. Ciesla SL, Trahan J, Wan WB, et al. Esterification of cidofovir with alkoxyalkanols

increases oral bioavailability and diminishes drug accumulation in kidney. Antiviral Res

2003; 59:163-171.

424. Hostetler KY. Alkoxyalkyl prodrugs of acyclic nucleoside phosphonates enhance oral

antiviral activity and reduce toxicity: current state of the art. Antiviral Res 2009; 82:A84-

A98.

425. Aldern KA, Ciesla SL, Winegarden KL, et al. Increased antiviral activity of 1-O-

hexadecyloxypropyl-[2-(14)C]cidofovir in MRC-5 human lung fibroblasts is explained

by unique cellular uptake and metabolism. Mol Pharmacol 2003; 63:678-681.

426. Xiong X, Smith JL, Chen MS. Effect of incorporation of cidofovir into DNA by human

cytomegalovirus DNA polymerase on DNA elongation. Antimicrob Agents Chemother

1997; 41:594-599.

427. Whitmer SL, Albarino C, Shepard SS, et al. Preliminary Evaluation of the Effect of

Investigational Ebola Virus Disease Treatments on Viral Genome Sequences. J Infect Dis

2016; 214:S333-S341.

428. Zhao Z, Martin C, Fan R, et al. Drug repurposing to target Ebola virus replication and

virulence using structural systems pharmacology. BMC Bioinformatics 2016; 17:10.1186.

429. Toth K, Spencer JF, Dhar D, et al. Hexadecyloxypropyl-cidofovir, CMX001, prevents

adenovirus-induced mortality in a permissive, immunosuppressed animal model. Proc

Natl Acad Sci U S A 2008; 105:7293-7297.

430. Zaitseva M, McCullough KT, Cruz S, et al. Postchallenge administration of brincidofovir

protects healthy and immune-deficient mice reconstituted with limited numbers of T cells

from lethal challenge with IHD-J-Luc vaccinia virus. J Virol 2015; 89:3295-3307.

431. Florescu DF, Kalil AC, Hewlett AL, et al. Administration of Brincidofovir and

Convalescent Plasma in a Patient With Ebola Virus Disease. Clin Infect Dis 2015;

61:969-973.

432. Painter W, Robertson A, Trost LC, et al. First pharmacokinetic and safety study in

humans of the novel lipid antiviral conjugate CMX001, a broad-spectrum oral drug active

against double-stranded DNA viruses. Antimicrob Agents Chemother 2012; 56:2726-

2734.

433. Yen HL. Current and novel antiviral strategies for influenza infection. Curr Opin Virol

2016; 18:126-134.

434. Jin Z, Smith LK, Rajwanshi VK, et al. The ambiguous base-pairing and high substrate

efficiency of T-705 (Favipiravir) Ribofuranosyl 5'-triphosphate towards influenza A virus

polymerase. PLoS One 2013; 8:e68347.

230

435. Westover JB, Sefing EJ, Bailey KW, et al. Low-dose ribavirin potentiates the antiviral

activity of favipiravir against hemorrhagic fever viruses. Antiviral Res 2016; 126:62-68.

436. Scharton D, Bailey KW, Vest Z, et al. Favipiravir (T-705) protects against peracute Rift

Valley fever virus infection and reduces delayed-onset neurologic disease observed with

ribavirin treatment. Antiviral Res 2014; 104:84-92.

437. Jin Z, Tucker K, Lin X, et al. Biochemical Evaluation of the Inhibition Properties of

Favipiravir and 2'-C-Methyl-Cytidine Triphosphates against Human and Mouse

Norovirus RNA Polymerases. Antimicrob Agents Chemother 2015; 59:7504-7516.

438. Delang L, Segura Guerrero N, Tas A, et al. Mutations in the chikungunya virus non-

structural proteins cause resistance to favipiravir (T-705), a broad-spectrum antiviral. J

Antimicrob Chemother 2014; 69:2770-2784.

439. Wang Y, Li G, Yuan S, et al. In Vitro Assessment of Combinations of Enterovirus

Inhibitors against Enterovirus 71. Antimicrob Agents Chemother 2016; 60:5357-5367.

440. Morrey JD, Taro BS, Siddharthan V, et al. Efficacy of orally administered T-705

pyrazine analog on lethal West Nile virus infection in rodents. Antiviral Res 2008;

80:377-379.

441. Madelain V, Guedj J, Mentre F, et al. Favipiravir Pharmacokinetics in Nonhuman

Primates and Insights for Future Efficacy Studies of Hemorrhagic Fever Viruses.

Antimicrob Agents Chemother 2017; 61: e01305-01316.

442. Kumagai Y, Murakawa Y, Hasunuma T, et al. Lack of effect of favipiravir, a novel

antiviral agent, on QT interval in healthy Japanese adults. Int J Clin Pharmacol Ther

2015; 53:866-874.

443. Schibler M, Vetter P, Cherpillod P, et al. Clinical features and viral kinetics in a rapidly

cured patient with Ebola virus disease: a case report. Lancet Infect Dis 2015; 15:1034-

1040.

444. Nguyen TH, Guedj J. Favipiravir pharmacokinetics in Ebola-Infected patients of the JIKI

trial reveals concentrations lower than targeted. PLoS Negl Trop Dis 2017; 11:e0005389.

445. Bai CQ, Mu JS, Kargbo D, et al. Clinical and Virological Characteristics of Ebola Virus

Disease Patients Treated With Favipiravir (T-705)-Sierra Leone, 2014. Clin Infect Dis

2016; 63:1288-1294.

446. Julander JG, Bantia S, Taubenheim BR, et al. BCX4430, a novel nucleoside analog,

effectively treats yellow fever in a Hamster model. Antimicrob Agents Chemother 2014;

58:6607-6614.

231

447. Julander JG, Siddharthan V, Evans J, et al. Efficacy of the broad-spectrum antiviral

compound BCX4430 against Zika virus in cell culture and in a mouse model. Antiviral

Res 2017; 137:14-22.

448. Taylor R, Kotian P, Warren T, et al. BCX4430 - A broad-spectrum antiviral adenosine

nucleoside analog under development for the treatment of Ebola virus disease. J Infect

Public Health 2016; 9:220-226.

449. Lo MK, Jordan R, Arvey A, et al. GS-5734 and its parent nucleoside analog inhibit Filo-,

Pneumo-, and Paramyxoviruses. Sci Rep 2017; 7:10.1038.

450. Jacobs M, Rodger A, Bell DJ, et al. Late Ebola virus relapse causing

meningoencephalitis: a case report. Lancet 2016; 388:498-503.

451. Dornemann J, Burzio C, Ronsse A, et al. First Newborn Baby to Receive Experimental

Therapies Survives Ebola Virus Disease. J Infect Dis 2017; 215:171-174.

452. Dowall SD, Bewley K, Watson RJ, et al. Antiviral Screening of Multiple Compounds

against Ebola Virus. Viruses 2016; 8:10.3390.

453. Liu G, Nash PJ, Johnson B, et al. A Sensitive in Vitro High-Throughput Screen To

Identify Pan-filoviral Replication Inhibitors Targeting the VP35-NP Interface. ACS Infect

Dis 2017; 3:190-198.

454. Madrid PB, Panchal RG, Warren TK, et al. Evaluation of Ebola Virus Inhibitors for Drug

Repurposing. ACS Infect Dis 2015; 1:317-326.

455. Jacome R, Becerra A, Ponce de Leon S, et al. Structural Analysis of Monomeric RNA-

Dependent Polymerases: Evolutionary and Therapeutic Implications. PLoS One 2015;

10:e0139001.

456. van Hemert FJ, Zaaijer HL, Berkhout B. In silico prediction of ebolavirus RNA

polymerase inhibition by specific combinations of approved nucleotide analogues. J Clin

Virol 2015; 73:89-94.

457. Lazcano A, Valverde V, Hernandez G, et al. On the early emergence of reverse

transcription: theoretical basis and experimental evidence. J Mol Evol 1992; 35:524-536.

458. Ricchetti M, Buc H. E. coli DNA polymerase I as a reverse transcriptase. Embo j 1993;

12:387-396.

459. Yang X, Smidansky ED, Maksimchuk KR, et al. Motif D of viral RNA-dependent RNA

polymerases determines efficiency and fidelity of nucleotide addition. Structure 2012;

20:1519-1527.

232

460. Doublie S, Tabor S, Long AM, et al. Crystal structure of a bacteriophage T7 DNA

replication complex at 2.2 A resolution. Nature 1998; 391:251-258.

461. Huang H, Chopra R, Verdine GL, et al. Structure of a covalently trapped catalytic

complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 1998;

282:1669-1675.

462. Alam I, Lee JH, Cho KJ, et al. Crystal structures of murine norovirus-1 RNA-dependent

RNA polymerase in complex with 2-thiouridine or ribavirin. Virology 2012; 426:143-

151.

463. Thompson AA, Albertini RA, Peersen OB. Stabilization of poliovirus polymerase by

NTP binding and fingers-thumb interactions. J Mol Biol 2007; 366:1459-1474.

464. Biswal BK, Cherney MM, Wang M, et al. Crystal structures of the RNA-dependent RNA

polymerase genotype 2a of hepatitis C virus reveal two conformations and suggest

mechanisms of inhibition by non-nucleoside inhibitors. J Biol Chem 2005; 280:18202-

18210.

465. Lohmann V, Korner F, Herian U, et al. Biochemical properties of hepatitis C virus NS5B

RNA-dependent RNA polymerase and identification of amino acid sequence motifs

essential for enzymatic activity. J Virol 1997; 71:8416-8428.

466. Vazquez AL, Alonso JM, Parra F. Mutation analysis of the GDD sequence motif of a

calicivirus RNA-dependent RNA polymerase. J Virol 2000; 74:3888-3891.

467. Bressanelli S, Tomei L, Roussel A, et al. Crystal structure of the RNA-dependent RNA

polymerase of hepatitis C virus. Proc Natl Acad Sci U S A 1999; 96:13034-13039.

468. Ng KK, Arnold JJ, Cameron CE. Structure-function relationships among RNA-dependent

RNA polymerases. Curr Top Microbiol Immunol 2008; 320:137-156.

469. Cerny J, Cerna Bolfikova B, Valdes JJ, et al. Evolution of tertiary structure of viral RNA

dependent polymerases. PLoS One 2014; 9:e96070.

470. Ferrer-Orta C, Arias A, Escarmis C, et al. A comparison of viral RNA-dependent RNA

polymerases. Curr Opin Struct Biol 2006; 16:27-34.

471. Gohara DW, Arnold JJ, Cameron CE. Poliovirus RNA-dependent RNA polymerase

(3Dpol): kinetic, thermodynamic, and structural analysis of ribonucleotide selection.

Biochemistry 2004; 43:5149-5158.

472. Garriga D, Ferrer-Orta C, Querol-Audi J, et al. Role of motif B loop in allosteric

regulation of RNA-dependent RNA polymerization activity. J Mol Biol 2013; 425:2279-

2287.

233

473. Joyce CM. Choosing the right sugar: how polymerases select a nucleotide substrate. Proc

Natl Acad Sci U S A 1997; 94:1619-1622.

474. Steitz TA. DNA polymerases: structural diversity and common mechanisms. J Biol Chem

1999; 274:17395-17398.

475. Wu Y, Lou Z, Miao Y, et al. Structures of EV71 RNA-dependent RNA polymerase in

complex with substrate and analogue provide a drug target against the hand-foot-and-

mouth disease pandemic in China. Protein Cell 2010; 1:491-500.

476. Choi KH, Groarke JM, Young DC, et al. The structure of the RNA-dependent RNA

polymerase from bovine viral diarrhea virus establishes the role of GTP in de novo

initiation. Proc Natl Acad Sci U S A 2004; 101:4425-4430.

477. Ferrer-Orta C, Arias A, Perez-Luque R, et al. Sequential structures provide insights into

the fidelity of RNA replication. Proc Natl Acad Sci U S A 2007; 104:9463-9468.

478. Schneidman-Duhovny D, Inbar Y, Nussinov R, et al. PatchDock and SymmDock: servers

for rigid and symmetric docking. Nucleic Acids Res 2005; 33:W363-367.

479. Mitsuya H, Weinhold KJ, Furman PA, et al. 3'-Azido-3'-deoxythymidine (BW A509U):

an antiviral agent that inhibits the infectivity and cytopathic effect of human T-

lymphotropic virus type III/lymphadenopathy-associated virus in vitro. Proc Natl Acad

Sci U S A 1985; 82:7096-7100.

480. Furman PA, Fyfe JA, St Clair MH, et al. Phosphorylation of 3'-azido-3'-deoxythymidine

and selective interaction of the 5'-triphosphate with human immunodeficiency virus

reverse transcriptase. Proc Natl Acad Sci U S A 1986; 83:8333-8337.

481. Larder BA, Kemp SD, Harrigan PR. Potential mechanism for sustained antiretroviral

efficacy of AZT-3TC combination therapy. Science 1995; 269:696-699.

482. WHO Guidelines Approved by the Guidelines Review Committee. In: Antiretroviral

Therapy for HIV Infection in Adults and Adolescents: Recommendations for a Public

Health Approach: 2010 Revision. Geneva: World Health Organization; 2010.

483. Parker WB, White EL, Shaddix SC, et al. Mechanism of inhibition of human

immunodeficiency virus type 1 reverse transcriptase and human DNA polymerases alpha,

beta, and gamma by the 5'-triphosphates of carbovir, 3'-azido-3'-deoxythymidine, 2',3'-

dideoxyguanosine and 3'-deoxythymidine. A novel RNA template for the evaluation of

antiretroviral drugs. J Biol Chem 1991; 266:1754-1762.

484. Konig H, Behr E, Lower J, et al. Azidothymidine triphosphate is an inhibitor of both

human immunodeficiency virus type 1 reverse transcriptase and DNA polymerase

gamma. Antimicrob Agents Chemother 1989; 33:2109-2114.

234

485. Desai VG, Lee T, Moland CL, et al. Effect of short-term exposure to zidovudine (AZT)

on the expression of mitochondria-related genes in skeletal muscle of neonatal mice.

Mitochondrion 2009; 9:9-16.

486. Dalakas MC, Illa I, Pezeshkpour GH, et al. Mitochondrial myopathy caused by long-term

zidovudine therapy. N Engl J Med 1990; 322:1098-1105.

487. Jaju M, Beard WA, Wilson SH. Human immunodeficiency virus type 1 reverse

transcriptase. 3'-Azidodeoxythymidine 5'-triphosphate inhibition indicates two-step

binding for template-primer. J Biol Chem 1995; 270:9740-9747.

488. Zhan X, Tan CK, Scott WA, et al. Catalytically distinct conformations of the

ribonuclease H of HIV-1 reverse transcriptase by substrate cleavage patterns and

inhibition by azidothymidylate and N-ethylmaleimide. Biochemistry 1994; 33:1366-1372.

489. Delviks-Frankenberry KA, Nikolenko GN, Barr R, et al. Mutations in human

immunodeficiency virus type 1 RNase H primer grip enhance 3'-azido-3'-deoxythymidine

resistance. J Virol 2007; 81:6837-6845.

490. Caliendo AM, Savara A, An D, et al. Effects of zidovudine-selected human

immunodeficiency virus type 1 reverse transcriptase amino acid substitutions on

processive DNA synthesis and viral replication. J Virol 1996; 70:2146-2153.

491. Sarafianos SG, Clark AD, Jr., Das K, et al. Structures of HIV-1 reverse transcriptase with

pre- and post-translocation AZTMP-terminated DNA. Embo j 2002; 21:6614-6624.

492. Boyer PL, Sarafianos SG, Clark PK, et al. Why do HIV-1 and HIV-2 use different

pathways to develop AZT resistance? PLoS Pathog 2006; 2:e10.

493. Galle PR, Theilmann L. Inhibition of hepatitis B virus polymerase-activity by various

agents. Transient expression of hepatitis B virus DNA in hepatoma cells as novel system

for evaluation of antiviral drugs. Drug Res 1990; 40:1380-1382.

494. Berk L, Schalm SW, Heijtink RA. Zidovudine inhibits hepatitis B virus replication.

Antiviral Res 1992; 19:111-118.

495. Macchi B, Faraoni I, Zhang J, et al. AZT inhibits the transmission of human T cell

leukaemia/lymphoma virus type I to adult peripheral blood mononuclear cells in vitro. J

Gen Virol 1997; 78:1007-1016.

496. Yvon-Groussin A, Mugnier P, Bertin P, et al. Efficacy of dideoxynucleosides against

human foamy virus and relationship to its reverse transcriptase amino acid sequence and

structure. J Virol 2001; 75:7184-7187.

497. Nam JH, Yu CH, Hwang KA, et al. Real-time RT-PCR of Hantaan virus RNA used for

the detection of virus response to antiviral drugs. Acta Virol 2008; 52:67-70.

235

498. Uckun FM, Venkatachalam TK, Erbeck D, et al. Zidampidine, an aryl phosphate

derivative of AZT: in vivo pharmacokinetics, metabolism, toxicity, and anti-viral efficacy

against hemorrhagic fever caused by Lassa virus. Bioorg Med Chem 2005; 13:3279-3288.

499. McCormick JB, Sasso DR, Palmer EL, et al. Morphological identification of the agent of

Korean haemorrhagic fever (Hantaan virus)as a member of the Bunyaviridae. Lancet

1982; 1:765-768.

500. Xiao SY, Liang M, Schmaljohn CS. Molecular and antigenic characterization of HV114,

a hantavirus isolated from a patient with haemorrhagic fever with renal syndrome in

China. J Gen Virol 1993; 74:1657-1659.

501. Kim JA, Kim WK, No JS, et al. Genetic Diversity and Reassortment of Hantaan Virus

Tripartite RNA Genomes in Nature, the Republic of Korea. PLoS Negl Trop Dis 2016;

10:e0004650.

502. Auperin DD, Sasso DR, McCormick JB. Nucleotide sequence of the glycoprotein gene

and intergenic region of the Lassa virus S genome RNA. Virology 1986; 154:155-167.

503. Lin MT, Chou YP, Hu TH, et al. Telbivudine and adefovir combination therapy for

patients with chronic lamivudine-resistant hepatitis B virus infections. Arch Virol 2014;

159:29-37.

504. Xie H, Voronkov M, Liotta DC, et al. Phosphatidyl-2',3'-dideoxy-3'-thiacytidine:

synthesis and antiviral activity in hepatitis B-and HIV-1-infected cells. Antiviral Res

1995; 28:113-120.

505. Coates JA, Cammack N, Jenkinson HJ, et al. (-)-2'-deoxy-3'-thiacytidine is a potent,

highly selective inhibitor of human immunodeficiency virus type 1 and type 2 replication

in vitro. Antimicrob Agents Chemother 1992; 36:733-739.

506. Hart GJ, Orr DC, Penn CR, et al. Effects of (-)-2'-deoxy-3'-thiacytidine (3TC) 5'-

triphosphate on human immunodeficiency virus reverse transcriptase and mammalian

DNA polymerases alpha, beta, and gamma. Antimicrob Agents Chemother 1992;

36:1688-1694.

507. Zhang Y, Song F, Gao Z, et al. Long-term exposure of mice to nucleoside analogues

disrupts mitochondrial DNA maintenance in cortical neurons. PLoS One 2014; 9:e85637.

508. Gray NM, Marr CL, Penn CR, et al. The intracellular phosphorylation of (-)-2'-deoxy-3'-

thiacytidine (3TC) and the incorporation of 3TC 5'-monophosphate into DNA by HIV-1

reverse transcriptase and human DNA polymerase gamma. Biochem Pharmacol 1995;

50:1043-1051.

236

509. Back NK, Nijhuis M, Keulen W, et al. Reduced replication of 3TC-resistant HIV-1

variants in primary cells due to a processivity defect of the reverse transcriptase enzyme.

Embo j 1996; 15:4040-4049.

510. Gao L, Hanson MN, Balakrishnan M, et al. Apparent defects in processive DNA

synthesis, strand transfer, and primer elongation of Met-184 mutants of HIV-1 reverse

transcriptase derive solely from a dNTP utilization defect. J Biol Chem 2008; 283:9196-

9205.

511. Oude Essink BB, Back NK, Berkhout B. Increased polymerase fidelity of the 3TC-

resistant variants of HIV-1 reverse transcriptase. Nucleic Acids Res 1997; 25:3212-3217.

512. Wei X, Liang C, Gotte M, et al. The M184V mutation in HIV-1 reverse transcriptase

reduces the restoration of wild-type replication by attenuated viruses. AIDS 2002;

16:2391-2398.

513. Sarafianos SG, Das K, Clark AD, Jr., et al. Lamivudine (3TC) resistance in HIV-1

reverse transcriptase involves steric hindrance with beta-branched amino acids. Proc Natl

Acad Sci U S A 1999; 96:10027-10032.

514. Severini A, Liu XY, Wilson JS, et al. Mechanism of inhibition of duck hepatitis B virus

polymerase by (-)-beta-L-2',3'-dideoxy-3'-thiacytidine. Antimicrob Agents Chemother

1995; 39:1430-1435.

515. Taylor GP, Hall SE, Navarrete S, et al. Effect of lamivudine on human T-cell leukemia

virus type 1 (HTLV-1) DNA copy number, T-cell phenotype, and anti-tax cytotoxic T-

cell frequency in patients with HTLV-1-associated myelopathy. J Virol 1999; 73:10289-

10295.

516. Das K, Xiong X, Yang H, et al. Molecular modeling and biochemical characterization

reveal the mechanism of hepatitis B virus polymerase resistance to lamivudine (3TC) and

emtricitabine (FTC). J Virol 2001; 75:4771-4779.

517. Urban S, Urban S, Fischer KP, et al. Efficient pyrophosphorolysis by a hepatitis B virus

polymerase may be a primer-unblocking mechanism. Proc Natl Acad Sci U S A 2001;

98:4984-4989.

518. Cong Y, Dyall J, Hart BJ. Evaluation of the Activity of Lamivudine and Zidovudine

against Ebola Virus. PLoS One 2016; 11:e0166318.

519. Hensley LE, Dyall J, Olinger GG, Jr., et al. Lack of effect of Lamivudine on ebola virus

replication. Emerg Infect Dis 2015; 21:550-552.

520. Zhao Y, Ren J, Harlos K, et al. Toremifene interacts with and destabilizes the Ebola virus

glycoprotein. Nature 2016; 535:169-172.

237

521. Suo Z, Johnson KA. Selective inhibition of HIV-1 reverse transcriptase by an antiviral

inhibitor, (R)-9-(2-Phosphonylmethoxypropyl)adenine. J Biol Chem 1998; 273:27250-

27258.

522. Nelson M, Portsmouth S, Stebbing J, et al. An open-label study of tenofovir in HIV-1

and Hepatitis B virus co-infected individuals. AIDS 2003; 17:F7-10.

523. Cassetti I, Madruga JV, Suleiman JM, et al. The safety and efficacy of tenofovir DF in

combination with lamivudine and efavirenz through 6 years in antiretroviral-naive HIV-

1-infected patients. HIV Clin Trials 2007; 8:164-172.

524. Johnson AA, Ray AS, Hanes J, et al. Toxicity of antiviral nucleoside analogs and the

human mitochondrial DNA polymerase. J Biol Chem 2001; 276:40847-40857.

525. Kulkarni R, Feng JY, Miller MD, et al. Dead-end complexes contribute to the synergistic

inhibition of HIV-1 RT by the combination of rilpivirine, emtricitabine, and tenofovir.

Antiviral Res 2014; 101:131-135.

526. Hachiya A, Kodama EN, Schuckmann MM, et al. K70Q adds high-level tenofovir

resistance to "Q151M complex" HIV reverse transcriptase through the enhanced

discrimination mechanism. PLoS One 2011; 6:e16242.

527. Chan PA, Huang A, Kantor R. Low prevalence of transmitted K65R and other tenofovir

resistance mutations across different HIV-1 subtypes: implications for pre-exposure

prophylaxis. J Int AIDS Soc 2012; 15:10.7448.

528. Jones SA, Murakami E, Delaney W, et al. Noncompetitive inhibition of hepatitis B virus

reverse transcriptase protein priming and DNA synthesis by the nucleoside analog

clevudine. Antimicrob Agents Chemother 2013; 57:4181-4189.

529. van Hemert FJ, Berkhout B, Zaaijer HL. Differential binding of tenofovir and adefovir to

reverse transcriptase of hepatitis B virus. PLoS One 2014; 9:e106324.

530. Oestereich L, Rieger T, Ludtke A, et al. Efficacy of Favipiravir Alone and in

Combination With Ribavirin in a Lethal, Immunocompetent Mouse Model of Lassa

Fever. J Infect Dis 2016; 213:934-938.

531. Staszewski S, Loveday C, Picazo JJ, et al. Safety and efficacy of lamivudine-zidovudine

combination therapy in zidovudine-experienced patients. A randomized controlled

comparison with zidovudine monotherapy. Lamivudine European HIV Working Group.

JAMA 1996; 276:111-117.

532. Xiao F, Fofana I, Thumann C, et al. Synergy of entry inhibitors with direct-acting

antivirals uncovers novel combinations for prevention and treatment of hepatitis C. Gut

2014; 64:483-494.

238

533. Chou TC. Theoretical basis, experimental design, and computerized simulation of

synergism and antagonism in drug combination studies. Pharmacol Rev 2006; 58:621-

681.

534. Hoenen T, Watt A, Mora A, et al. Modeling The Lifecycle Of Ebola Virus Under

Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic

Minigenomes. J Vis Exp 2014; 91:52381.

535. Hoenen T, Feldmann H. Reverse genetics systems as tools for the development of novel

therapies against filoviruses. Expert Rev Anti Infect Ther 2014; 12:1253-1263.

536. de Avila AI, Gallego I, Soria ME, et al. Lethal Mutagenesis of Hepatitis C Virus Induced

by Favipiravir. PLoS One 2016; 11:e0164691.

537. Bermejo M, Lopez-Huertas MR, Garcia-Perez J, et al. Dasatinib inhibits HIV-1

replication through the interference of SAMHD1 phosphorylation in CD4+ T cells.

Biochem Pharmacol 2016; 106:30-45.

538. Di Mascio M, Srinivasula S, Bhattacharjee A, et al. Antiretroviral tissue kinetics: in vivo

imaging using positron emission tomography. Antimicrob Agents Chemother 2009;

53:4086-4095.

539. Harmon B, Campbell N, Ratner L. Role of Abl kinase and the Wave2 signaling complex

in HIV-1 entry at a post-hemifusion step. PLoS Pathog 2010; 6:e1000956.

540. Lund N, Olsson ML, Ramkumar S, et al. The human P(k) histo-blood group antigen

provides protection against HIV-1 infection. Blood 2009; 113:4980-4991.

541. Bokaei PB, Ma XZ, Sakac D, et al. HIV-1 integration is inhibited by stimulation of the

VPAC2 neuroendocrine receptor. Virology 2007; 362:38-49.

542. Yamamoto N, Tanaka C, Wu Y, et al. Analysis of human immunodeficiency virus type 1

integration by using a specific, sensitive and quantitative assay based on real-time

polymerase chain reaction. Virus Genes 2006; 32:105-113.

543. O'Doherty U, Swiggard WJ, Jeyakumar D, et al. A sensitive, quantitative assay for

human immunodeficiency virus type 1 integration. J Virol 2002; 76:10942-10950.

544. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T)

method. Nat Protoc 2008; 3:1101-1108.

545. Kim M, Binnington B, Sakac D, et al. Comparison of detection methods for cell surface

globotriaosylceramide. J Immunol Methods 2011; 371:48-60.

239

546. Vermeire J, Naessens E, Vanderstraeten H, et al. Quantification of reverse transcriptase

activity by real-time PCR as a fast and accurate method for titration of HIV, lenti- and

retroviral vectors. PLoS One 2012; 7:e50859.

547. Akari H, Uchiyama T, Fukumori T, et al. Pseudotyping human immunodeficiency virus

type 1 by vesicular stomatitis virus G protein does not reduce the cell-dependent

requirement of vif for optimal infectivity: functional difference between Vif and Nef. J

Gen Virol 1999; 80 ( Pt 11):2945-2949.

548. Guendel I, Agbottah ET, Kehn-Hall K, et al. Inhibition of human immunodeficiency

virus type-1 by cdk inhibitors. AIDS Res Ther 2010; 7:7.

549. Puertas MC, Massanella M, Llibre JM, et al. Intensification of a raltegravir-based

regimen with maraviroc in early HIV-1 infection. Aids 2014; 28:325-334.

550. Wei DG, Chiang V, Fyne E, et al. Histone deacetylase inhibitor romidepsin induces HIV

expression in CD4 T cells from patients on suppressive antiretroviral therapy at

concentrations achieved by clinical dosing. PLoS Pathog 2014; 10:e1004071.

551. Desimmie BA, Schrijvers R, Demeulemeester J, et al. LEDGINs inhibit late stage HIV-1

replication by modulating integrase multimerization in the virions. Retrovirology 2013;

10:57.

552. Shan L, Xing S, Yang HC, et al. Unique characteristics of histone deacetylase inhibitors

in reactivation of latent HIV-1 in Bcl-2-transduced primary resting CD4+ T cells. J

Antimicrob Chemother 2014; 69:28-33.

553. Shah NP, Guilhot F, Cortes JE, et al. Long-term outcome with dasatinib after imatinib

failure in chronic-phase chronic myeloid leukemia: follow-up of phase 3 study. Blood

2014.

554. Pogliaghi M, Papagno L, Lambert S, et al. The tyrosine kinase inhibitor Dasatinib blocks

in-vitro HIV-1 production by primary CD4+ T cells from HIV-1 infected patients. AIDS

2014; 28:278-281.

555. McCarthy SD, Jung D, Sakac D, et al. c-Src and Pyk2 Protein Tyrosine Kinases Play

Protective Roles in Early HIV-1 Infection of CD4+ T-Cell Lines. J Acquir Immune Defic

Syndr 2014; 66:118-126.

556. Pizzato M, Erlwein O, Bonsall D, et al. A one-step SYBR Green I-based product-

enhanced reverse transcriptase assay for the quantitation of retroviruses in cell culture

supernatants. J Virol Methods 2009; 156:1-7.

557. Bain J, Plater L, Elliott M, et al. The selectivity of protein kinase inhibitors: a further

update. Biochem J 2007; 408:297-315.

240

558. Harvey R, Hehir KM, Smith AE, et al. pp60c-src variants containing lesions that affect

phosphorylation at tyrosines 416 and 527. Mol Cell Biol 1989; 9:3647-3656.

559. Collins M, Tremblay M, Chapman N, et al. The T cell receptor-mediated phosphorylation

of Pyk2 tyrosines 402 and 580 occurs via a distinct mechanism than other receptor

systems. J Leukoc Biol 2010; 87:691-701.

560. Mehla R, Bivalkar-Mehla S, Zhang R, et al. Bryostatin modulates latent HIV-1 infection

via PKC and AMPK signaling but inhibits acute infection in a receptor independent

manner. PLoS One 2010; 5:e11160.

561. Tyler T. Once-daily dasatinib for treatment of patients with chronic myeloid leukemia.

Ann Pharmacother 2009; 43:920-927.

562. Bartelt RR, Cruz-Orcutt N, Collins M, et al. Comparison of T cell receptor-induced

proximal signaling and downstream functions in immortalized and primary T cells. PLoS

One 2009; 4:e5430.

563. Rato S, Maia S, Brito PM, et al. Novel HIV-1 knockdown targets identified by an

enriched kinases/phosphatases shRNA library using a long-term iterative screen in Jurkat

T-cells. PLoS One 2010; 5:e9276.

564. Zhou H, Xu M, Huang Q, et al. Genome-scale RNAi screen for host factors required for

HIV replication. Cell Host Microbe 2008; 4:495-504.

565. Hatano H, Strain MC, Scherzer R, et al. Increase in 2-long terminal repeat circles and

decrease in D-dimer after raltegravir intensification in patients with treated HIV

infection: a randomized, placebo-controlled trial. J Infect Dis 2013; 208:1436-1442.

566. Buzon MJ, Massanella M, Llibre JM, et al. HIV-1 replication and immune dynamics are

affected by raltegravir intensification of HAART-suppressed subjects. Nat Med 2010;

16:460-465.

567. Hazuda DJ, Felock P, Witmer M, et al. Inhibitors of strand transfer that prevent

integration and inhibit HIV-1 replication in cells. Science 2000; 287:646-650.

568. World Health Organization. Ebola Situation Report – 16 December 2015. 2015.

http://apps.who.int/ebola/en/current-situation/ebola-situation-report-13-december-2015

569. Wong G, Audet J, Fernando L, et al. Immunization with vesicular stomatitis virus

vaccine expressing the Ebola glycoprotein provides sustained long-term protection in

rodents. Vaccine 2014; 32:5722-5729.

570. Wong KK, Perdue CL, Malia J, et al. Supportive Care of the First 2 Ebola Virus Disease

Patients at the Monrovia Medical Unit. Clin Infect Dis 2015; 61:e47-51.

241

571. Florescu DF, Keck MA. Development of CMX001 (Brincidofovir) for the treatment of

serious diseases or conditions caused by dsDNA viruses. Expert Rev Anti Infect Ther

2014; 12:1171-1178.

572. Muhlberger E, Weik M, Volchkov VE, et al. Comparison of the transcription and

replication strategies of marburg virus and Ebola virus by using artificial replication

systems. J Virol 1999; 73:2333-2342.

573. Phanuphak N, Ananworanich J, Teeratakulpisarn N, et al. A 72-week randomized study

of the safety and efficacy of a stavudine to zidovudine switch at 24 weeks compared to

zidovudine or tenofovir disoproxil fumarate when given with lamivudine and nevirapine.

Antivir Ther 2012; 17:1521-1531.

574. World Health Organization. 18th WHO Model List of Essential Medicines (April 2013).

2013. http://www.who.int/medicines/publications/essentialmedicines/en/

575. Ahmed Ouameur A, Marty R, Neault JF, et al. AZT binds RNA at multiple sites. DNA

Cell Biol 2004; 23:783-788.

576. Cardenas WB. Evasion of the interferon-mediated antiviral response by filoviruses.

Viruses 2010; 2:262-282.

577. Xu W, Edwards MR, Borek DM, et al. Ebola virus VP24 targets a unique NLS binding

site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated

STAT1. Cell Host Microbe 2014; 16:187-200.

578. Fabozzi G, Nabel CS, Dolan MA, et al. Ebolavirus proteins suppress the effects of small

interfering RNA by direct interaction with the mammalian RNA interference pathway. J

Virol 2011; 85:2512-2523.

579. Chang TH, Kubota T, Matsuoka M, et al. Ebola Zaire virus blocks type I interferon

production by exploiting the host SUMO modification machinery. PLoS Pathog 2009;

5:e1000493.

580. Qiu X, Wong G, Fernando L, et al. mAbs and Ad-vectored IFN-alpha therapy rescue

Ebola-infected nonhuman primates when administered after the detection of viremia and

symptoms. Sci Transl Med 2013; 5:207ra143.

581. Qiu X, Wong G, Fernando L, et al. Monoclonal antibodies combined with adenovirus-

vectored interferon significantly extend the treatment window in Ebola virus-infected

guinea pigs. J Virol 2013; 87:7754-7757.

582. Ebihara H, Theriault S, Neumann G, et al. In vitro and in vivo characterization of

recombinant Ebola viruses expressing enhanced green fluorescent protein. J Infect Dis

2007; 196:S313-322.

242

583. Hoenen T, Jung S, Herwig A, et al. Both matrix proteins of Ebola virus contribute to the

regulation of viral genome replication and transcription. Virology 2010; 403:56-66.

584. Mehrotra S, Sharma B, Joshi S, et al. Essential role for the Mnk pathway in the inhibitory

effects of type I interferons on myeloproliferative neoplasm (MPN) precursors. J Biol

Chem 2013; 288:23814-23822.

585. Watt A, Moukambi F, Banadyga L, et al. A novel life cycle modeling system for Ebola

virus shows a genome length-dependent role of VP24 in virus infectivity. J Virol 2014;

88:10511-10524.

586. Johansen LM, DeWald LE, Shoemaker CJ, et al. A screen of approved drugs and

molecular probes identifies therapeutics with anti-Ebola virus activity. Sci Transl Med

2015; 7:290ra289.

587. Lok AS, Lai CL, Leung N, et al. Long-term safety of lamivudine treatment in patients

with chronic hepatitis B. Gastroenterol 2003; 125:1714-1722.

588. Baeten JM, Donnell D, Mugo NR, et al. Single-agent tenofovir versus combination

emtricitabine plus tenofovir for pre-exposure prophylaxis for HIV-1 acquisition: an

update of data from a randomised, double-blind, phase 3 trial. Lancet Infect Dis 2014;

14:1055-1064.

589. Thoulouze MI, Sol-Foulon N, Blanchet F, et al. Human immunodeficiency virus type-1

infection impairs the formation of the immunological synapse. Immunity 2006; 24:547-

561.

590. Uitdehaag JCM, Verkaar F, Alwan H, et al. A guide to picking the most selective kinase

inhibitor tool compounds for pharmacological validation of drug targets. Br J Pharmacol

2012; 166:858-876.

591. Shi H, Zhang CJ, Chen GY, et al. Cell-based proteome profiling of potential dasatinib

targets by use of affinity-based probes. J Am Chem Soc 2012; 134:3001-3014.

592. Abraham RT, Weiss A. Jurkat T cells and development of the T-cell receptor signalling

paradigm. Nat Rev Immunol 2004; 4:301-308.

593. Bronevetsky Y, Villarino AV, Eisley CJ, et al. T cell activation induces proteasomal

degradation of Argonaute and rapid remodeling of the microRNA repertoire. J Exp Med

2013; 210:417-432.

594. Kinter A, McNally J, Riggin L, et al. Suppression of HIV-specific T cell activity by

lymph node CD25+ regulatory T cells from HIV-infected individuals. Proc Natl Acad Sci

U S A 2007; 104:3390-3395.

243

595. Pallikkuth S, Sharkey M, Babic DZ, et al. Peripheral T Follicular Helper Cells Are the

Major HIV Reservoir within Central Memory CD4 T Cells in Peripheral Blood from

Chronically HIV-Infected Individuals on Combination Antiretroviral Therapy. J Virol

2015; 90:2718-2728.

596. Blom N, Sicheritz-Ponten T, Gupta R, et al. Prediction of post-translational glycosylation

and phosphorylation of proteins from the amino acid sequence. Proteomics 2004; 4:1633-

1649.

597. Chen P, Li F, Xu Z, et al. Expression and distribution of Src in the nucleus of myocytes

in cardiac hypertrophy. Int J Mol Med 2013; 32:165-173.

598. Cooper A, Garcia M, Petrovas C, et al. HIV-1 causes CD4 cell death through DNA-

dependent protein kinase during viral integration. Nature 2013; 498:376-379.

599. Patel M, Philip V, Fazel F, et al. Human immunodeficiency virus infection and chronic

myeloid leukemia. Leuk Res 2012; 36:1334-1338.

600. Schlaberg R, Fisher JG, Flamm MJ, et al. Chronic myeloid leukemia and HIV-infection.

Leuk Lymphoma 2008; 49:1155-1160.

601. Chung WM, Smith JC, Weil LM, et al. Active Tracing and Monitoring of Contacts

Associated With the First Cluster of Ebola in the United States. Ann Intern Med 2015;

163:164-173.

602. Tippin TK, Morrison ME, Brundage TM, et al. Brincidofovir Is Not a Substrate for the

Human Organic Anion Transporter 1: A Mechanistic Explanation for the Lack of

Nephrotoxicity Observed in Clinical Studies. Ther Drug Monit 2016; 38:777-786.

603. M'Kada H, Munteanu M, Perazzo H, et al. What are the best reference values for a

normal serum alanine transaminase activity (ALT)? Impact on the presumed prevalence

of drug induced liver injury (DILI). Regul Toxicol Pharmacol 2011; 60:290-295.

604. World Health Organization. Ebola Situation Report, 20 June 2017 2017.

http://apps.who.int/ebola/ebola-situation-reports

605. García M, Cooper A, Shi W, et al. Productive Replication of Ebola Virus Is Regulated by

the c-Abl1 Tyrosine Kinase. Sci Transl Med 2012; 4:123ra124.

606. Bekerman E, Neveu G, Shulla A, et al. Anticancer kinase inhibitors impair intracellular

viral trafficking and exert broad-spectrum antiviral effects. J Clin Invest 2017; 127:1338-

1352.

607. Schweitzer CJ, Jagadish T, Haverland N, et al. Proteomic analysis of early HIV-1

nucleoprotein complexes. J Proteome Res 2013; 12:559-572.

244

608. Raghavendra NK, Shkriabai N, Graham R, et al. Identification of host proteins associated

with HIV-1 preintegration complexes isolated from infected CD4+ cells. Retrovirology

2010; 7:10.1186.

609. Fujisaka Y, Onozawa Y, Kurata T, et al. First report of the safety, tolerability, and

pharmacokinetics of the Src kinase inhibitor saracatinib (AZD0530) in Japanese patients

with advanced solid tumours. Invest New Drugs 2013; 31:108-114.

610. Haile WB, Gavegnano C, Tao S, et al. The Janus kinase inhibitor ruxolitinib reduces HIV

replication in human macrophages and ameliorates HIV encephalitis in a murine model.

Neurobiol Dis 2016.

611. Bermejo M, Lopez-Huertas MR, Garcia-Perez J, et al. Dasatinib inhibits HIV-1

replication through the interference of SAMHD1 phosphorylation in CD4+ T cells.

Biochem Pharmacol 2016.

612. Gavegnano C, Detorio M, Montero C, et al. Ruxolitinib and tofacitinib are potent and

selective inhibitors of HIV-1 replication and virus reactivation in vitro. Antimicrob

Agents Chemother 2014; 58:1977-1986.

613. Haile WB, Gavegnano C, Tao S, et al. The Janus kinase inhibitor ruxolitinib reduces HIV

replication in human macrophages and ameliorates HIV encephalitis in a murine model.

Neurobiol Dis 2016; 92:137-143.

614. Alfson KJ, Worwa G, Carrion R, Jr., et al. Determination and Therapeutic Exploitation of

Ebola Virus Spontaneous Mutation Frequency. J Virol 2015; 90:2345-2355.

615. Sun W, He S, Martinez-Romero C, et al. Synergistic drug combination effectively blocks

Ebola virus infection. Antiviral Res 2017; 137:165-172.

245

Copyright Acknowledgments

The author acknowledges the following publications, from which previously published work has

been reproduced in this thesis, with permission:

McCarthy, S.D., Jung, D., Sakac, D., and Branch, D.R. 2014. c-SRC and Pyk2 protein tyrosine

kinases play protective roles in early HIV-1 infection of CD4+ T-cell lines. JAIDS, 66(2):

118-26.

McCarthy, S.D., Sakac, D., Neschadim, A., and Branch, D.R. 2016. c-SRC protein tyrosine

kinase regulates early HIV-1 infection post-entry. AIDS, 30(6): 849-58.

McCarthy, S.D., Majchrzak-Kita, B., Racine, T., Kozlowski, H.N., Baker, D.P., Hoenen, T.,

Kobinger, G.P., Fish, E.N., and Branch, D.R. 2016. A Rapid Screening Assay Identifies

Monotherapy with Interferon-ß and Combination Therapies with Nucleoside Analogues

as Effective Inhibitors of Ebola Virus. PLoS NTD, 10(1):e0004364.

4. GARP, UNAIDS. Global AIDS Update. 2016.

http://www.unaids.org/en/resources/documents/2016/Global-AIDS-update-2016

5. World Health Organization. Ebola Situation Report, 10 June 2016. 2016.

http://apps.who.int/ebola/ebola-situation-reports

43. Faria NR, Rambaut A, Suchard MA, et al. HIV epidemiology. The early spread and

epidemic ignition of HIV-1 in human populations. Science 2014; 346:56-61.

44. Disease NIoAaI, National Institutes Health. How HIV Causes AIDS. 2004.

45. Suzuki Y, Suzuki Y. Gene Regulatable Lentiviral Vector System. In: Viral Gene

Therapy. Edited by Xu K. Rijeka: InTech; 2011. pp. 10.5772.

53. Coiras M, Lopez-Huertas MR, Perez-Olmeda M, et al. Understanding HIV-1 latency

provides clues for the eradication of long-term reservoirs. Nat Rev Microbiol 2009;

7:798-812.

169. Wu P, Nielsen TE, Clausen MH. Small-molecule kinase inhibitors: an analysis of FDA-

approved drugs. Drug Discov Today 2016; 21:5-10.

246

170. Wu P, Nielsen TE, Clausen MH. FDA-approved small-molecule kinase inhibitors. Trends

Pharmacol Sci 2015; 36:422-439.

198. Okada M. Regulation of the SRC family kinases by Csk. Int J Biol Sci 2012; 8:1385-

1397.

199. Engen JR, Wales TE, Hochrein JM, et al. Structure and dynamic regulation of Src-family

kinases. Cell Mol Life Sci 2008; 65:3058-3073.

221. Fackler OT, Alcover A, Schwartz O. Modulation of the immunological synapse: a key to

HIV-1 pathogenesis? Nat Rev Immunol 2007; 7:310-317.

328. Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: in command and control

of cell motility. Nat Rev Mol Cell Biol 2005; 6:56-68.

329. Walkiewicz KW, Girault JA, Arold ST. How to awaken your nanomachines: Site-specific

activation of focal adhesion kinases through ligand interactions. Prog Biophys Mol Biol

2015; 119:60-71.

368. Artimo P, Jonnalagedda M, Arnold K, et al. ExPASy: SIB bioinformatics resource portal.

Nucleic Acids Res 2012; 40:W597-603.

455. Jacome R, Becerra A, Ponce de Leon S, et al. Structural Analysis of Monomeric RNA-

Dependent Polymerases: Evolutionary and Therapeutic Implications. PLoS One 2015;

10:e0139001.

456. van Hemert FJ, Zaaijer HL, Berkhout B. In silico prediction of ebolavirus RNA

polymerase inhibition by specific combinations of approved nucleotide analogues. J Clin

Virol 2015; 73:89-94.

562. Bartelt RR, Cruz-Orcutt N, Collins M, et al. Comparison of T cell receptor-induced

proximal signaling and downstream functions in immortalized and primary T cells. PLoS

One 2009; 4:e5430.