HIV-1 and Sickle Cell Disease - Howard University Home and SCD 2011.pdfHIV-1 and Sickle Cell Disease Sergei Nekhai, Ph.D. NHLBI, NIH NIGMS, NIH The Office of Research on Minority Health,

HIV-1 and Sickle Cell Disease

Sergei Nekhai, Ph.D.

NHLBI, NIH

NIGMS, NIH

The Office of Research on

Minority Health, NIH

NIAID, NIH

Center for Sickle Cell Disease,

Howard University

Sickle Cell Disease

• Affects about 100,000 people in US

• Occurs in about 1 of 500 African

American births (~1000 per yr)

• Occurs in 1 of 1,000 to 1,400 Hispanic

American births.

HbS Polymer

(Tavassoli, M)

Patophysiology

• Sickle erythrocyte membrane damage

• Adhesion of sickle erythrocytes to

endothelium

• Hemolytic vasculopathy

• Chronic inflammatory response

• Chronic hypoxia

• Splenic atrophy

• 2/73 SCD pts (2.7%) exposed to >1300 units

unscreened blood 1978-84 became HIV +

• 8/101 pts (7.9%) Tx’d 1978-88 became HTLV-1 +

• P = 0.15 Pearson chi square; 0.20 Fisher exact

• “Our analysis suggests a low risk for HIV

infection in SCD pts transfused before HIV

donor screening”

Journal of Infectious Diseases 1990;162:743-745

• 5 university centers

• 8/18 HIV+ Hb SS pts (44.4%) long-term non-

progressors (none on HAART)

• 5/36 HIV+ control pts (13.9%) long-term non-

progressors

• P = 0.013 Pearson chi square, 0.020, Fisher exact

Am Journal of Hematology 1998;59:199-207

J National Med Assoc 2003;95:813-817

Mechanisms?

• Hypoxic response

• Inflammatory response

• Hypocholesterolemia

• Others?

CYTOPLASM

ENTRY

REVERSE

TRANSCRIPTION

NUCLEUS

INTEGRATION

TRANSCRIPTIONTRANSLATION

BUDDING ASSEMBLY

MATURATION

UNCOATING

VIRAL

PROTEINS

DENDRITIC

CELL

CELL

FACTORS

CD4,

CXCR4,

CCR5,

D6

DC-SIGN

Pol,

DBR1

Int, Vpu,

PARP1

Tat, LTR,

CDK9, cycT1,

Spt5, CDK2

SPLICING

RNA TRANSPORT

PROCESSING

CyPA

Rev,

DHS, hRIP, SAM68

Gag, env

LIP5

Vif,

Cul5-E3

HIV-1 Life Cycle

Nekhai and Jerebtsova,

Curr.Opin.Mol. Therapy, 2006

TRIM5a

APOBEC3G

Vpu Tetherin

RPII

CTDTat

HEXIM1

Cyclin T1CDK9 S175P

T186-P

Cyclin T1

CDK9

Cyclin T1

CDK9

PPPPP

TatPP1

Tat

PP1

NUCLEUS

CYTOPLASM

7SK RNA

Model of Regulation of HIV-1 Transcription by PP1

T186-P

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

SS1 SS2 SS3 AA1 AA2

Rel

. lu

cife

rase

act

ivit

y a

dju

sted

to t

he

cell

num

ber

PBMCs without HIV-1

PBMCs infected with HIV-1

Replication of HIV-1 Luciferase Virus is Reduced in

PBMCs Isolated from Sickle Cell Patients and

Cultured at 3% Oxygen

Hypothesis for SCD Protection from HIV-1

• SCD causes recurrent ischemia/hypoxia which

may affect HIV-1

• SCD causes decreased expression of hepcidin

which may slower rates of HIV-1 replication

• Lower cholesterol levels at SCD may affect HIV-

1 entry (lipid rafts) and transcription (SREBP-

2/THII-I – Dr. Hildreth)

• Activation of heme oxygenase inhibits HIV-1 in

macrophages

Hypoxia and HIV-1 Transcription

• Physiological concentration of O2 is 3-6% in theperipheral tissues (Meyron-Holtz et al., Science2004)

1 % oxygen 21 % oxygen

HIV-1 is Suppressed at 3% O2

0

200

400

600

800

1000

1200

1400

1600

1800

0 1 2 3 4 5

U1, 21% O2, - TNF

U1, 3% O2, -TNF

U1, 21% O2 +TNF

U1, 3% O2 + TNF

Days

p24

, p

g/m

l

A

BU1 Monocytes

HIV-1 Luc - + - +

21% O23% O2

1 2 3 4

Lu

cif

era

se

ac

tivit

y,

% o

f c

on

tro

l

0

25

50

75

100

125CEM

THP-1

PBMC

Charles et al, J Cell Physiol. 2009

tubulin

B CDK9

WB

O2, % 21 3

cyclin T1

a-tubulinWB

O2, % 21 3

WB

IgG

CDK9

O2, % 21 21 3

IP: IgG CDK9

WB

Cyclin T1O2, % 21 21 3

IP: IgG CDK9

CCTDa

CTDo(32P)

O2, % 21 21 3

IP: IgG CDK9

IgG

CTDa

CTDo(32P)

O2, % 21 3 21

IP: cyclin T1 IgG

rCD

K9

D 21% O2 IC50=0.17 mM

3% O2 IC50=3 mM

ARC,mM

0

20

40

60

80

100

120

140

10-8 10-7 10-6 10-5 10-4

HIV

-1 T

ran

scri

pti

on

, %

of

un

trea

ted

con

trol

A

CDK9 Activity is Modulated at 3% O2

Charles et al, J Cell Physiol. 2009

Hypoxia and Protein Phosphatase-1

PP1 is repressed during hypoxia (Taylor et al.,PNAS 2000) through increased interaction withNIPP1 (Comerford et al, J Cell Physiol. 2006)

Comerford et al, J Cell Physiol. 2006

PP1

NIPP1 192PKRKRKNSRVTFS204

PKA

NIPP1 192PKRKRKNSRVTFS204

P P

PP1

A

B

0

100

200

300

400

500

600

700

a-NIPP1 IgG

21% O2

3% O2

Ph

osp

ha

tase A

ctivity,

Art

bitra

ry U

nits

IP: NIPP

WB:NIPP1 NIPP1*

C

1 2 3 4

IP: IgG NIPP1

atm

osO

2

ph

ys O

2

atm

osO

2

atm

osO

2 100

33

0

20

40

60

80

100

120

21% O2 3% O2

Ph

osp

ha

tase A

ctivity,

% o

f A

tmo

sO

2co

ntr

ol

Hypoxia Deregulates Protein Phosphatase-1

0 5 10 20 40 60 0 5 10 20 40 60

Time Intervals of Trypsin Treatment

21% O23% O2

PP1

PP1 Activity Is Lower in Cells Cultured at

3% Oxygen

0

1000

2000

3000

4000

5000

6000

293T 84-31 Y724 84-31

pA/RATA

CEM

32P

re

lea

se

, re

l. u

nits

21%O2

3%O2

PP1 Targets CDK9 Ser 175

Ammosova et al., PLOS One 2011

A

S175A

WT

Mock

IP a-FLAG

(32P)

Coomassie

B

CDK9

CDK9C

TL

C

TLE

TLE

TL

C

13

2

E Spot #3

DSpot #1 and #2 WT CDK9

WT CDK9 S175A

Ser175

Copyright ©2008 Ferrata Storti Foundation

Kemna, E. H.J.M. et al. Haematologica 2008;93:90-97

Iron Uptake and Recycling

Ferroportin Q248H

• Fpn encodes a multiple transmembrane domain

protein, involved in iron export

• Pathogenic mutations: associated with iron

loading

• Q248H (c.DNA 744G T)

• Q248H unique to Africans and is associated with a

tendency to iron loading

Effect of Ferroportin and Hepcidin on HIV-1

B HIV-1 LTR HIV-1 LTR+Tat

CD4

FPN WT

Tra

nsa

cri

pti

on

Ac

tiva

tio

n,

(Fo

ld)

1 2 0

5

10

15

20

25

30

35

40

A

FPN

a-tubulin

CD4 + -

FPN - +

FPN

C

0

20

40

60

80

100

120

140

160

No

. o

f E

ven

ts

anti-c-mycIgG

10-2

Red Fluorescence

10-1 100 101 102 103 104

Xu et al., Retrovirology 2010

0

500

1000

1500

2000

2500

RT

act

ivit

y (

cou

nts

)

CD4+

T cells

Primary

Monocytes

FAC - + - + Hepcid - - + +

RT

act

ivit

y (

cou

nts

)

0

2000

4000

6000

8000

10000

FAC - + - + Hepcid - - + +

WB: anti-a-tubulin

WB: anti-myc Fpn

Tubulin

0 0.01 0.03 0.1 0 0.01 0.03 0.1 0 0.01 0.03 0.1 Hepcidin (mM)

GFPFpn WT Fpn C326YFpn Q248H

1 2 3 4 5 6 7 8 9 10 11 12 13

A

Sensitivity of Ferroportin Q248H Mutant to Hepcidin

B Ferroportin-EGFP

0

20

40

60

80

100

120

0 0.05 0.1 0.15

Hepcidin, uM

% o

f u

ntr

ea

ted

co

ntr

ol

FPN WT

Q248H

C326Y

0

100

200

300

400

500

600FPN WT

FPN Q248H Heterozygote

Fpn Q248H Homozygote

HIV-1 Luc - + + +

Iron+AA - - + +

Hepcidin - - - +

Lu

cife

rase a

ctivity,

arb

itra

ry u

nits

A

B

Ferroportin Q248H Mutant Restricts HIV-1 Replication

0

0.5

1

1.5

2

2.5

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07

Hepcidin, uM

WT

Q248H

C326Y

Lucifera

se a

ctivity,

arb

itra

ry u

nits

Conclusions/Directions• HIV-1 replication is slower in SCD-derived

PBMCs; in cell cultured at 3% O2 or cells

expressing ferroportin

• Activities of host cell CDK9 and PP1 are

modulated at 3% O2

• Hepcidin induces HIV-1, and Fpn 248, insensitive

to hepcidin, protects against HIV-1

• In future, analyze PP1 activity and hepcidin

expression an in SCD

• Use iPSC technology to study Fpn Q248

• Analyze effect of cholersterol to HIV-1 in SCD

AcknowledgementsVictor Gordeuk Howard University,

Tatiana Ammosova Center for Sickle

Sharroya Charles Cell Disease

Altreisha Foster

Min Xu,

Denitra Breuer

Jamie Rotimie

Zufan Debebe

Yuri Obukhiov

Fatah Kashanchi George Mason University, VA

Mathieu Bollen Catholic University, Leuven, Belgium

Marina Jerebtsova Children’s National Medical Center

Patricio Ray