John Short Leeds Dissertation

IMM SHORT

Viral Evasion of the

Interferon Gateway

John A. L. Short 200114360 Dr. Andrew Macdonald

SUBMITTED IN ACCORDANCE WITH THE REQUIREMENTS FOR THE DEGREE OF BSC IN MICROBIOLOGY WITH

IMMUNOLOGY (IND), UNIVERSITY OF LEEDS UNDERGRADUATE SCHOOL OF BIOLOGICAL SCIENCES

14th APRIL 2008

THE CANDIDATE CONFIRMS THAT THE WORK IS SUBMITTED

IN ACCORDANCE WITH THE DECLARATION OF ACADEMIC INTEGRITY SIGNED BY THE CANDIDATE AT THE START OF

THE ACADEMIC YEAR

14th April 2008 Viral Evasion of the Interferon Gateway John A L Short

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Contents

ABSTRACT 1

1. INTRODUCTION 2

1.1. The Virus-Host Dynamic 2

1.2. The Interplay between Innate Immunity and Virus Infection 3

1.3. Extracellular Antiviral Components 4

1.4. Intracellular Antiviral Components: The Interferon Gateway 5

Toll-Like Receptors 5

Viral Recognition in the Cytosol 7

1.5. The Antiviral State 9

Interferon Stimulated Genes 11

1.6. Aims 13

2. VIRAL INTERFERNCE OF IFN-α/β EXPRESSION 14

2.1. Viral Interference of Initial Pattern Recognition 14

2.2. Viral Interference with the TLR Signalling Pathways 16

2.3. Viral interference of the RIG-I / MDA5 Signalling Pathways 18

2.4. Viral Interference of the IFN-α/β signal transduction pathways 21

TBK-1: The vital link 21

Targeting the IFN-α/β transcription Factors 24

IRF-3/ IRF-7 degradation 25

Viral Disruption of the IRF-3/CBP/p300 complex 29

3. VIRAL INTERFERENCE OF THE JAK/STAT PATHWAY 32

3.1. IFNAR receptor disruption 32

3.2. Viral inhibition of JAK kinase Activity 33

3.3. STAT Protein Sequestration 36

3.4. Viral Induction of STAT Protein Degradation 39

3.5. Viral Inhibition of STAT trafficking 40

3.6. ISGF3 Promoter Interference 42


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4. VIRAL INTERFERENCE OF ISGs 43

4.1. PKR 43

PKR domain interaction 44

PKR degradation 47

Viral Targeting of Phosphorylated eIF2α 48

4.2. RNase L 49

4.3. APOBECs 50

4.4. ADAR-1 51

4.5. Tetherin 51

4.6. PML 51

5. DISCUSSION 53

5.1. Nature of Viral Inhibition 54

5.2. Comparing RNA and DNA Viral Evasion Strategies 55

Viral Evasion and effect on lifestyle 57

Genus and strain variation 58

5.3. Antiviral Therapies 59

Additional Therapeutic Opportunities 61

5.4. Conclusion 62

6. ACKNOWLEDGEMENTS 62

7. REFERENCES 63

8. APPENDICES 75

8.1. Abbreviations 75


1

Abstract

Viruses and their hosts since the dawn of time have been battling for supremacy. In

recent years the Interferon Gateway encompassing interferon alpha and beta (IFN-

α/β) expression, signalling and antiviral responses, has been uncovered. IFN-α/β are

cytokines that co-ordinate the innate and adaptive immune responses to eliminate

virus infections from the host. Interferon Stimulated Gene products such as PKR can

destroy viral and cellular mRNAs to limit viral replication, but can also initiate

apoptosis if the cell is overwhelmed. In order to survive, RNA and DNA viruses have

evolved viral evasion proteins that are able to target all aspects of the Interferon

Gateway through a variety of sophisticated mechanisms. Viral evasion proteins can

encode cellular domains, directly neutralising the gateway, hijacking cellular

pathways or degrading antiviral components. High mutational rates of viral

replication ensure that viruses will continue to adapt to our defences, but equally the

viral evasion proteins are novel drug targets for eliminating or managing virus

infections and can be subverted for the treatment of autoimmune disorders.


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1. Introduction

1.1. The Virus-Host Dynamic

Viruses and their hosts have a dynamic relationship, constantly evolving strategies to

outwit the other in a battle for survival. Viruses have developed various strategies for

evading and subverting the defence mechanisms of the host for their own needs.

Viruses cause significant human morbidity and mortality, such as annual influenza

epidemics that are estimated to cause three to five million cases of severe illness and

between 250,000 and 500,000 deaths per year globally [1]. The development of

vaccines and antivirals against viruses such as Human Immunodeficiency Virus (HIV)

and influenza is expensive and prone to failure due to their inherent adaptability of

the viruses to the host defences and antiviral therapies [2, 3].

The host has two main pathways for eliminating virus infections; the innate immune

response and the adaptive immune response. The innate response is the first line of

defence, recognising general features of pathogens by pattern recognition receptors

(PRRs) which detect pathogen associated molecular patterns (PAMPS) [4]. This

initial response is rapid and aims to either clear the infection or hold it at bay until an

adaptive response is mounted. The adaptive response is critical in eliminating

pathogens that have evolved specific features that avoid initial recognition.

Historically the innate immune response has been considered to be simple and

unimportant compared to the adaptive response which has been the main focus of

immunological research. Whilst the adaptive immune system is capable of

eliminating specific virus infections, there has only recently been an awareness of

how complex and critically important the innate response is for curbing viral

replication and initiating the adaptive response. Research in this area is fragmented

with very few overall “big picture” analyses of the viral evasion and subversion

strategies of innate immunity. The innate immune system consists of a variety of

intracellular and extracellular components that are able to, either by themselves or in

conjunction with the adaptive immune response, eliminate pathogens.


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1.2. The Interplay between Innate Immunity and Virus infection

Viruses have specific cellular tropisms that are dictated by the expression of specific

virus receptors. These are located either on the virus capsid or within the lipid of

enveloped viruses [5]. Extracellular and intracellular arms of the innate immune

system have evolved to prevent virus infection of host cells, to eliminate the virus

after infection and to impair viral replication and infection of uninfected cells before

the adaptive immune system has a chance to respond (Figure 1).

Fig. 1. The Innate Immune System Matrix. When the virus penetrates the external barrier, it disseminates via the

bloodstream or through tissues until it encounters its target cell, presenting various ligands that activate the

extracellular and intracellular arms of the innate immune system (see text). Green dashed arrows indicate the target

of cytokines produced. Pink dashed arrows show the target of IFN-α/β produced. Modified from [4, 6-8].


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The innate immune responses are interconnected and feed into the adaptive

response via the action of cytokines. These are protein chemical messengers

secreted by cells in response to viral ligands [6]. They can act in an auto-, para and

endocrine manner to generate an immune response. The innate immune system is

able to co-ordinate the adaptive response and vice versa. The mass orchestration of

the innate immune system is necessary to generate a sufficient response to

neutralise the virus.

1.3. Extracellular Antiviral components

Viruses are prevented from invading their target cell by the mechanical barriers of the

skin and mucosal immune system. Epithelial cells of the mucosal immune system

and keratinocytes produce microbial peptides called defensins that are capable of

neutralising enveloped viruses [9]. Defensins inhibit Lentivirus replication and are

chemoattractants for T-cells.

Upon breaching this barrier, virus particles can activate the complement system.

This consists of three cascades catalysed by proteases that form protein cleavage

products and complexes which are deposited on the viral envelope or virus particles

in serum. Antibody-antigen complexes and viral oligosaccharides are ligands for the

Classical and Mannin Binding Lectin pathways respectively, whereas the Alternative

Pathway is activated by the spontaneous breakdown and disposition of complement

[10]. Complement can lyse enveloped viruses through the formation of the

membrane attack complex via all three pathways, or it can facilitate virus clearance

by cells that express complement receptors such as macrophages [11].

Virus particles trigger inflammation through activated complement and the secretion

of cytokines from infected cells and leukocytes that cause inflammation. Inflammation

is a key antiviral response that reduces viral replication and recruits immune effecter

cells to the site of infection [12]. Pro-inflammatory cytokines cause the local

vasodilation of blood vessels increasing blood flow. This reduces viral replication by

raising the local temperature and improving access for innate and adaptive immune

effecter cells. Other cytokines are able to chemoattract and modulate the activity of

immune effecter cells. Macrophages and plasmoidal dendritic cells (pDCs) are able

to phagocytose infectious virus particles and proteins derived from lysed cells and

expose viral antigens to the adaptive immune system [12, 13].


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Recently it has been found that virus infected and neighbouring uninfected cells are

able to generate intracellular antiviral resistance by the action of interferons (IFN) [14].

IFNs are a class of cytokine that act as the “gatekeepers” of innate and adaptive

immunity, exhibiting a global influence on the action of antiviral extracellular and

intracellular immune responses. IFNs orchestrate these responses to reduce or

prevent virus replication and dissemination until the immune effecter cells eliminate

the virus and infected cells. The importance of the Interferon Gateway has been

demonstrated by the vast array of strategies that viruses have evolved for evading

and subverting this immune defence system, which will be the main subject of this

review.

1.4. Intracellular Antiviral components: The Interferon Gateway

The transcription of Type I alpha and beta interferons (IFN-α/β) is the major form of

control on the activation of immune responses [4]. IFN-α/β help to mediate the

activation and coordination of immune effecter cells (Figure 1). They are critical for

generating antiviral resistance in both infected and uninfected cells by increasing the

expression of Interferon Stimulated Genes (ISGs). IFN-α is produced predominately

in pDCs, whereas IFN-β is produced in most nucleated cells [15]. The regulation of

IFN-α/β expression is crucial as unwarranted antiviral responses could lead to cell

damage and apoptosis. Over the last decade our understanding of the activation and

regulation of IFN-α/β has increased significantly.

Toll-Like Receptors

Many viruses exploit the endocytic system during their life-cycle. This is a major

transportation hub, where endosome transport vesicles are used both for the initial

infection of the cell by a virus particle and also for egress of virions containing newly

replicated genomes [16]. To prevent virus subversion of this key organelle the host

has evolved a class of sentinel PRRs that reside in the endocytic system. These Toll-

like receptors (TLRs) recognise pathogen structural components and viral nucleic

acids. For example TLR3 detects dsRNA, TLR9 senses viral unmethylated CpG

dsDNA and TLR7/8 recognise viral ssRNA [8, 17]. Although TLR4 is located on the

plasma membrane and does not recognise viral nucleic acid, it is able to detect viral

envelope proteins and transduce signals through a similar cascade as TLR3 [18].


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Detection of viral PAMPs by TLRs triggers various recently described intracellular

signalling cascades (Figure 2).

Fig. 2. The TLR signal cascade. TLRs are activated by their appropriate ligand (see text) and dimerise. This results

in the recruitment of signalling complexes which initiate activation of a signalling cascade. This leads to the activation

of the IFN-α/β transcription factors (see text). The transcription factors dimerise with their appropriate partner if

necessary and enter the nucleus, binding to host cell DNA at the IFN-α/β promoter regions. The transcription factors

described assemble on the promoter regions of IFN-α or IFN-β and initiate transcription of the genes. *TLR4 is

localised to the cell membrane. Green dashed arrows represent phosphorylation. Modified from [4, 8, 19].

TLRs reside as monomers in the endosome membrane that dimerise upon binding to

viral ligands [20]. They recruit the TIR domain-containing adaptor inducing IFN-β

(TRIF) and Myeloid differentiation factor 88 (MyD88) adaptor proteins that initiate

signal transduction cascades through the recruitment of further adaptor proteins and


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protein kinases. Recruitment of the signal kinase platforms TANK binding kinase 1

(TBK-1), Tumour necrosis factor receptor-associated factor 6 (TRAF6) and Inhibitor

of NF-κB activator (IKKβ) activates the IFN-α/β transcription factors critical for

induction of IFN-α/β gene expression. Interferon Regulatory Factor (IRF) 3, IRF-7, c-

Jun and Activating Transcription Factor 2 (AFT-2) are localised in the cytosol until

they are phosphorylated by their respective platforms, allowing them to dimerise with

their appropriate partner and translocate to the nucleus. Recruitment of the IKKβ

complex allows the activation of Nuclear Factor κB (NF-κB) by phosphorylating the

inhibitor of the IFN-β transcription factor NF-κB (IκBα). Dissociation and subsequent

degradation of the phosphorylated inhibitor allows NF-κB to translocate to the

nucleus [21]. The induction of IFN-α/β expression occurs by the binding of the

appropriate transcription factors to their respective promoter (Figure 2).

Viral recognition in the Cytosol

Many viruses utilise the cytosol, either for genome replication or intracellular

transport. Unsurprisingly, the host has evolved cytosolic detectors of viral nucleic

acids. These sentinel proteins are analogous to the TLR proteins localised in cellular

membranes. Melanoma differentiation associated gene 5 (MDA5) and Retinoic acid

inducible gene I (RIG-I) contain RNA helicase domains that detect viral RNA [22].

The cytosolic sensors initiate a signal transduction pathway culminating in the

transcription of IFN-α/β (Figure 3).


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Fig. 3. RIG-I and MDA5 Viral nucleic acid recognition pathways. The Repressor Domains (RD) and the helicase

domains of RIG-I and LGP2 inactivate the CARD domain of RIG-I. When ssRNA with a 5’ triphosphate cap or

dsRNA binds to the helicase domain, this enables Tripartite Motif 25 (TRIM25, a ubiquitin ligase) to commence

ubiquitnation (Ub) of the CARD domain of RIG-I and dissociation of LGP2. Activated RIG-I and MDA5 (by binding of

dsRNA to its helicase domain) allows the CARD domains of both to interact with IPS-1 adaptor protein. Signal

transduction pathways are activated by IPS-1, whereby the transcription factors IRF-3, 7 and NF-κB are activated

through phosphorylation. C-Jun and ATF-2 are activated via an as yet undefined mechanism. These enter the

nucleus, binding to the promoter regions of IFN-α/β, resulting in their transcription. In this pathway TRAF6 interacts

with the Fas Associated death domain (FADD). Green arrows represent phosphorylation. Dashed lines represent

undefined pathway. Modified from [4, 8, 19].

MDA5 and RIG-I bind to long nucleic acids, a feature of viral dsRNA produced from

complementary annealing of ssRNA from RNA viruses or from convergent


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transcription from DNA viruses [23]. Host cell RNA is much shorter in length, which

allows convenient sensor discrimination between viral and host cell RNA. Recent

studies demonstrate that RIG-I also binds to ssRNA with a 5’ triphosphate group.

Host ssRNA (mRNA) is post-transcriptionally modified with a 5’ cap structure, which

prevents detection by RIG-I and subsequent auto-activation of the innate immune

response to host nucleic acids [24]. The activated sensors initiate signal transduction

via adaptor protein domains called Caspase Activation and Recruitment Domain

(CARDs) that interact with homologous CARDs found on downstream signalling

components [25]. IFN-β promoter stimulator 1 (IPS-1) is a key intermediary of MDA5

and RIG-I signalling, activating the IFN-α/β transcription factors via the recruitment of

homologous signal kinase platforms as described for TLRs. This leads to their

translocation to the nucleus inducing IFN-α/β expression which subsequently

mediates the generation of cellular antiviral resistance.

1.5. The Antiviral State

Expression of IFN-α/β in response to presence of viral ligands leads to the generation

of antiviral resistance by activation of IFN-α/β signal induction pathways and

subsequent action of Interferon Stimulated Genes (ISGs). ISGs are expressed at low

levels in nucleated cells so that the cell has some degree of response to a viral

infection [4]. IFN-α/β signals by binding to the Type I Interferon receptor (IFNAR) in

an autocrine and paracrine manner, activating the JAK/STAT signal transduction

pathway leading to ISG transcription [14]. This generates an antiviral state in virus

infected and non infected cells in order to prevent and reduce further viral infection

and replication (Figure 4).


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Fig. 4. The JAK/STAT pathway. IFN-α/β binds to the type I IFN receptors (IFNAR) which then form a heterodimer.

They are associated with the Tyk2 and Jak1 kinases that activate a signal transduction pathway which leads to the

expression of numerous IFN-stimulated genes (see text). Green dashed arrows indicate phosphorylation. Modified

from [4, 8, 14]

IFNAR1 and 2 when bound to IFN-α/β dimerise, activating the receptor associated

Janus Kinases (JAK), Janus kinase 1 (Jak1) and Tyrosine Kinase 2 (Tyk2). These

phosphorylate the ISG transcription factor proteins, Signal Transducers and

Activators of Transcription (STAT) that subsequently dimerise and translocate to the

nucleus [26]. Upon entering the nucleus the STAT heterodimer interacts and binds

to IRF-9, forming the ISG transcription factor complex IFN-stimulated gene factor 3

(ISGF3). ISGF3 binds to the IFN-stimulated response element (ISRE), inducing

transcription of ISGs that are capable of countering virus infection [27].


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Interferon Stimulated Genes

Several ISGs have been identified with dsRNA-dependent Protein Kinase R (PKR)

gene product being the best characterised. PKR is a serine threonine kinase that is

activated by binding to dsRNA. PKR has two domains, an N-terminal regulatory

dsRNA binding domain and the C-terminal catalytic domain that contains conserved

motifs for acting on various transcription and translation factors [28].

PKR

PKR

PKR

dsRNA

P

P

eIF2a

P

eIF2aIkBa

P

IkBa

Fig. 5. The activation of PKR. Upon binding to viral dsRNA of at least 50bps long, PKR undergoes a conformational

change and autophosphoylates forming a dimer, which exposes the catalytic domain. The active PKR then

phosphorylates eIF2α and IKKβ (see text). Green dashed arrows indicate phosphorylation. Modified from [4, 29].

Elongation initiation factor 2 subunit alpha (eIF2α) is a critical translation cofactor

required for the recruitment of initiator Methionine Transfer RNA to ribosomes to form

the translation pre-initiation complex. The nucleotide exchange factor eIF2B

mediates the recycling of eIF2α, releasing it from the complex so that it can

participate in the translation of other mRNAs. PKR phosphorylates eIF2α enabling it

to irreversibly bind to the nucleotide exchange factor eIF2B. As eIF2B activity is

inhibited by phosphorylated eIF2α, this “freezes” eIF2α in the complex preventing it

from initiating future translational events [30] . This prevents the ribosomal translation

of cellular and viral proteins, ultimately blocking viral replication in the cell.

PKR also mediates virus clearance by interacting with other components of the

innate immune system. PKR can phosphorylate the IKKβ complex (Figure 2 and 3).

The IKKβ complex then phosphorylates the NF-κB inhibitor as previously described,

activating NF-κB Furthermore, PKR can activate cellular apoptosis pathways to

destroy the cell before the virus can fully replicate and assemble [31].


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2’-5’ OAS (2'-5' Oligoadenylate Synthetase) is an enzyme that upon binding to viral

dsRNA, uses adenosine triphosphate (ATP) as a substrate to catalyse the synthesis

of adenosine oligomers linked by phosphodiester bonds in a 2’ to 5’ configuration.

These strongly interact and activate endoribonuclease L (RNase L) [32]. RNase L

then cleaves cellular and viral ssRNA and mRNA, which inhibits translation of viral

proteins [33]. Sufficient degradation of cellular RNA can cause activation of apoptosis

pathways that could cause lysis of the cell destroying the virus before it has had time

to sufficiently replicate and assemble.

Mx proteins are highly conserved Guanine Tyrosine Phosphatases that interfere with

virus replication. They impede viral transcription by inhibiting the localisation and

activity of viral polymerases [34].

Adenosine deaminase RNA 1 (ADAR-1) deaminates dsRNA viral replication

intermediates. It replaces adenosines with inosine which causes the dsRNA to

unwind disrupting viral replication [14].

Promyelocytic leukaemia (PML) nuclear bodies are heterogeneous in size and

composition, and contain the IFN inducible protein PML and other IFN-α/β inducible

proteins, such as Sp100. They play roles in transcriptional responses to stress and

may regulate chromatin structure and promoter accessibility. Overexpression of

certain isoforms of PML impairs the replication of both RNA and DNA viruses,

although the details of their involvement remain to be determined [35].

Cellular restriction factors Apolipoprotein B mRNA editing enzyme–catalytic

polypeptide-like (APOBEC) and Tripartite motif-5 alpha (TRIM5α) are enzymatic

antivirals. The mechanism of action of APOBECs involves both cytidine deamination

and subsequent mutation of the viral genome and inhibition of reverse transcriptase

activities (if applicable) [36]. TRIM5α shows species-specific antiviral activity against

Retroviruses. TRIM5α interacts with incoming viral capsids and forms a complex

which signals its localisation for destruction to the proteasome [37].

To reinforce the immune response, IFN-α/β upregulates the gene expression of

detectors of viral nucleic acids and proteins, including the TLRs, RIG-I and MDA5.

This generates a positive feedback loop that consequently enables increased

detection of viral PAMPs and subsequent increased ISG expression. The feed back

loop subsequently amplifies the intensity of the cellular antiviral state so that the rate

of virus replication and infection is greatly diminished.


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IFN-α/β has profound immunomodulatory influence on adaptive immune cells and

antigen presentation cells, by upregulating class I Major Histocompatibility Complex

(MHC) molecules and components of the antigen-presenting machinery [38]. This

mechanism helps to counter the frequently observed downregulation of class I MHC

associated with specific viruses, which is beyond the scope of this review. IFN-α/β

also help to activate Natural Killer cells by complex processes including the

upregulation of perforin and granzymes [39].

1.6. Aims

Viruses have evolved various strategies to actively evade and subvert the host innate

immune response at all steps. Many viruses have adapted by expressing viral

proteins that act as “keys”, modulating the Interferon Gateway by “locking” or

inhibiting multiple levels to enable viral replication and assembly in the cell. By

focusing on how viruses are able to achieve this, the aim is to evaluate the current

understanding of viral evasion strategies. Using this “big picture” analysis, novel drug

targets could be elucidated that would aid the host innate immune response to

eliminate the virus, preventing viral pathology before the adaptive response kicks in

e.g. with influenza infections or in the event the virus is able to overcome the

adaptive response e.g. infection with HIV.


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2. Viral Interference of IFN-α/β Expression

To prevent the generation of the antiviral state in cells and the global co-ordination of

immune responses (see Introduction), viruses have evolved viral proteins that inhibit

the expression of IFN-α/β. As IFN-α/β are the key mediators of the Interferon

Gateway, inhibition of IFN-α/β expression would lead to a major loss of the antiviral

response capability of the innate immune system. Viruses have evolved evasion

proteins to target the cellular recognition of viral PAMPs, where if the cell cannot

detect infection, downstream signalling responses will remain inactive and viral

replication and assembly will continue undisturbed.

2.1. Viral Interference of Initial pattern Recognition

The TLRs, MDA5 and RIG-I detect specific viral nucleic acids (see Introduction) and

initiate downstream signal transduction pathways that culminate in the expression of

IFN-α/β. Viruses can express proteins that bind to viral dsRNA at specific sites

impeding an interaction with the PRRs (Table 1)

Table 1

Virus Nucleic acid Viral Evasion Protein

Influenza A Negative sense ssRNA NS1

Ebola Virus Negative sense ssRNA VP35

BVDV Positive sense ssRNA Erns

Rotavirus dsRNA Sigma3

HSV dsDNA Us11

EBV dsDNA EB2

VACV dsDNA E3L

Viruses encoding dsRNA binding proteins. (see text) Modified from [40-45].

Influenza A is a highly infectious respiratory tract infection causing significant

pathology. The Influenza A NS1 protein binds to both dsRNA and ssRNA, although

the affinity for dsRNA is greater. The viral RNA is recognised by specific amino acid

motifs within NS1 N-terminus. Mutagenesis studies of the NS1 N-terminus indicate

that Arg 38 and Lys 41 are necessary to mediate dsRNA binding. Structural

analyses imply that Arg 38 binds electrostatically to the dsRNA, and that Lys 41

contributes to affinity of binding [40].

Ebola Virus infections are incurable, causing haemorrhagic fever with upwards of

90% mortality [46]. In comparison Rotaviruses are relatively non-pathogenic [47].


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Ebola VP35 and the Rotavirus Sigma3 proteins perform the same function as NS1,

sharing significant sequence homology within their dsRNA binding domains (Figure

6).

Influenza A virus NS1 L-R-R-D-Q-K-S-L-R-G-R

Zaire Ebola Virus VP35 P-R-A-C-Q-K-S-L-R-P-V

Rotavirus Sigma3 i) K-G-R-A-Y-R- K

Rotavirus Sigma3 ii) K- L-K-T-V- R-K

304314

240234

297291

36 46

Fig. 6. Sequence Homology between NS1, VP35 and Sigma3 viral evasion proteins. Rotavirus sigma3 has two

RNA binding domains i) and ii). Red coloured amino acids (a. a.) represent those found to be critical in RNA binding

in NS1. Blue circled amino acids represent the key R-X-X-X-K RNA binding signal motif (where X stands for any a.

a.). Black lines indicate amino acids shared between proteins. Modified from [40-42].

NS1, Sigma3 and VP35 RNA binding domains share an R-X-X-X-K consensus

sequence [40-42]. This indicates that under selective pressure from the Interferon

Gateway, by convergent evolution they have targeted a similar sequence in dsRNA

that enables its efficient sequestration. Uniquely among RNA viruses to date, Bovine

Viral Diarrhoea Virus (BVDV) secretes the viral glycoprotein termed Erns with dsRNA

binding and RNase activities [45]. This has the cumulative effect of both binding and

degrading viral dsRNA to prevent activation of the antiviral response.

Herpes Simplex Virus (HSV) and Epstein-Barr Virus (EBV) are from the

Herpesviridae family which generally cause lifelong persistent infections, where

immune responses are not generated despite the presence of viral PAMPs. The C-

terminus of the Us11 protein of HSV and the nuclear protein EB2 of EBV both

contain a homologous Arg-X-Pro tripeptide repeat [43, 44]. Through mutational

studies these repeats have been shown to be critical for binding to dsRNA, thus

preventing intracellular viral recognition. This contributes to persistence within cells.

Vaccinia Virus (VACV) is a large, complex, dsDNA virus that encodes two dsRNA-

binding proteins p20 and p25 from the E3L gene [48]. VACV infection cause mild,

often asymptomatic pathology, but is mainly of interest due to its structure similar to

that of the related to Variola Virus, the causative agent of smallpox [49]. Smallpox

causes 30% mortality in humans, but was eradicated in 1979 after a comprehensive

http://en.wikipedia.org/wiki/DNA


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global vaccination programme [50]. Viral dsRNA is sequestered by the C-termini of

p20 and p25.

Infection of cells with mutants of the above viral evasion proteins resulted in

increased viral sensitivity to IFN-α/β with a pronounced reduction in the rate of viral

replication [45, 51-55]. The diverse array of viruses that encode dsRNA binding

proteins reflects the vital importance of disrupting the Interferon Gateway to prevent

the generation of the antiviral response.

2.2. Viral Interference of the TLR Signalling Pathways

Viruses that infect via the endocytic pathway expose viral PAMPs to TLR3 and 4

signalling pathways. TLR3 detects dsRNA and TLR4 detects viral glycoproteins

present in the cell membrane of enveloped viruses [56]. Certain viruses have

evolved to target two key adaptor proteins that facilitate TLR signalling, TRIF and

MyD88; disabling the pathway at that point and inhibiting IFN-β expression (Figure 7).


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Fig. 7. Viral interference of TLR3 and TLR4 signalling pathways of IFN-β induction. Viral proteins are able to

interfere with specific steps in the pathway; preventing the activation of the IFN-β transcription factors (see text).

TLR4 can recruit TRIF and MyD88 via Translocating chain-associating membrane (TRAM) and Myelin and

lymphocyte protein (MAL) respectively. VACV A46 binds to TIR domains present in TRIF, TLR4, MAL and MyD88.

However, A46 only partially inhibits NF-κB activation. A52 binds TRAF6, blocking NF-κB activation. The proteins of

RV and BVDV bind to TBK-1 IRF-3 phosphorylation (see text). Green dashed arrows show phosphorylation.

Diagram modified from [4, 8, 57-59].

A46 is the principal viral evasion protein of VACV. It binds to the Toll/Interleukin-1

Receptor (TIR) domains present in the TLR adaptor proteins TRIF and MyD88 [57].

The TIR domain is a protein interaction module that mediates multiple protein-protein

interactions. The formation of TIR-TIR interactions between the TLRs and their

respective signalling molecules mediates downstream signalling and IFN-β

production (Figure 7) [58]. The interaction with A46 obstructs TRIF recruitment to

TLR3 and TLR4 upon binding to viral PAMP and disables initial signal transduction.

As a result IRF-3 is not phosphorylated and remains inactive in the cytosol.


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A46 also binds to the TIR domains present within the TLR4 associated adaptor

proteins Myelin and lymphocyte (MAL) and MyD88. However, cell transfection

assays demonstrate that TLR3/4 TRIF and MyD88 mediated NF-κB activation is only

partially inhibited by A46 [57]. In order to prevent NF-κB activation, VACV encodes a

highly effective NF-κB inhibitor called A52. A52 binds to the TRAF6 complex, potently

inhibiting NF-κB activation [60]. The dual effect of A46 and A52 results in the reduced

expression of IFN-β and ISGs.

The Hepatitis C Virus (HCV) is a positive sense ssRNA virus that causes persistent

liver inflammation that is often asymptomatic, but which eventually results in cirrhosis

and liver cancer. The HCV NS3/4A serine protease contributes to persistence by

disrupting the TLR3 signal pathway (Figure 3) [59]. The viral NS3/4A protease

cleaves TRIF at Cys 372, causing the loss of the TBK-1 recruitment domain from the

TRIF protein [61]. With the loss of this domain, IRF-3 is not phosphorylated,

reducing the cellular expression of IFN-β by ~75% compared to NS3/4A deficient

HCV, promoting the persistent infections seen in pathology [59].

2.3. Viral interference of the RIG-I / MDA5 Signalling Pathways

Viruses that replicate in the cytosol or transgress through it to bud from the cell have

the potential to activate the interferon gateway via the cytosolic sentinels MDA5 and

RIG-I (see Introduction). Evolution has provided a similar evasion strategy from

evading TLRs, and many viruses target the cytosolic sensors preventing them from

initiating the signal transduction pathways necessary for generating the cellular

antiviral state (Figure 8).

http://en.wikipedia.org/wiki/Liver

http://en.wikipedia.org/wiki/Cirrhosis

http://en.wikipedia.org/wiki/Hepatocellular_carcinoma


19

Fig. 8. Viral evasion of the Cytosolic sensor PRRs. The V and C proteins of Paramyxoviruses bind to helicase

domains of MDA5 and RIG-I respectively. Influenza A NS1 targets CARD domains of IPS-1 and RIG-I. NS3/4A

targets membrane anchor domain of IPS-1 [62-64].

Paramyxoviruses are enveloped negative sense ssRNA viruses that include

Parainfluenza Virus (PIV), Measles Virus (MeV), Mumps Virus (MuV) and

Respiratory Syncytial Virus (RSV). These viruses cause significant disease in

humans as well as animals. Although vaccines are available for MeV and MuV,

these viruses combined caused 745,000 global deaths in 2001. Infections with RSV

and PIV can result in life threatening bronchitis and pneumonia. The above viruses

encode a “V” protein that interacts with the helicase domain of MDA5, inhibiting

activation by preventing the interaction with dsRNA. The C-termini of V proteins are

highly conserved between Paramyxoviruses that target humans and those that target

other species, such as avian Newcastle Disease Virus and Sendai Virus (SeV). They

contain a Cys rich C-terminus that binds to the MDA5 helicase domain, sequestering

it from dsRNA [65]. That thirteen viruses of different genera within the

Paramyxoviridae family have evolved to target MDA5 is indicative of the particular

importance of MDA5 inhibition to viruses of this family [66].

All Paramyxoviruses generate 5'-triphosphorylated ssRNA in the cytosol during RNA

virus replication which can potentially be recognised by RIG-I. Some

Paramyxoviruses such as SeV encode an additional C protein that is able to bind to

RIG-I preventing induction of IFN-β [67]. The C and V proteins are encoded by

separate alternate open reading frames (ORFs), which both overlap that of the P

protein (Figure 9).

http://www.kidshealth.org/parent/infections/lung/rsv.html


20

Fig. 9. Sendai Virus genetic structure. The C′, C, Y1 and Y2 proteins, collectively called C proteins, are encoded

by an overlapping open reading frame of the upstream regions of the P and V mRNAs with multiple translational start

codons and a common termination codon. V and C proteins from different genera can have similar or different

functions (see text). Paramyxoviruses also encode L and W proteins. Modified from [67, 68].

Infection with the SeV C protein in cells containing RIG-I showed markedly

decreased IFN-β production compared to that of cells with MDA5 or the dominant-

negative form of RIG-I (whose N-terminal CARD domains were deleted), indicating

that RIG-I is the predominant sensor. The C protein was found to bind to the RIG-I

helicase domain and not MDA5 indicating that this pathway was the most important

inducing IFN-β expression [64]. Other paramyxovirus V proteins such as measles do

not have this activity [66], indicating that they have other methods of immune evasion

or a divergence in the life-cycle of these viruses where one needs to evade both RIG-

I/MDA5 and one that does not.

The NS1 protein of Influenza A interacts with RIG-I, determined by co-

immunopreciptation experiments [63]. Influenza viral ssRNA can be detected by

RIG-I (see Introduction) leading to the expression of viral countermeasures. The

NS1 protein disrupts cell signalling by forming a trimeric complex with RIG-I and the

key signalling intermediary IPS-1 by an as yet undetermined mechanism this

prevents down-stream signalling and IRF-3 phosphorylation.

As mentioned previously, HCV encodes a viral protease, NS3/4A. This protease also

targets IPS-1 by cleaving at Cys 508 located in the IPS-1 C-terminus [69] (Figure 10).


21

Fig. 10. IPS-1 Structure. The Proline rich region (Pro) interacts with TRAF6. IPS-1 CARD domain interacts with

homologous MDA5 and ubiquitinated RIG-I CARD domains. NS3/4A cleaves IPS-1 at Cys 508 in the C-terminus

located just before the mitochondrial bound transmembrane region (TM). Modified from [69].

Cleavage of IPS-1 results in the redistribution of IPS-1 from the mitochondria to the

cytosol. There it cannot recruit the TRAF6 complex to the mitochondrial membrane

which is essential for the activation of downstream pathways to activate NF-κB [62].

Secondly it cannot activate the TBK-1 complex as IRF-3 and IRF-7 phosphorylation

was abolished [69]. HCV used a similar strategy to counter the TLR3 pathway by

cleaving TRIF with NS3/4A, illustrating a common approach by HCV to disabling key

antiviral signalling pathways.

2.4. Viral Interference of the IFN-α/β signal transduction pathways

Many viruses disrupt the downstream signalling pathways that are common to the

TLRs and the cytosolic sensors by targeting TBK-1 and the IFN-α/β transcription

factors. In doing so they are able to inhibit more than one pathway thus further

enhancing viral replication.

TBK-1: The vital link

The TBK-1 complex is the underlying platform linking recognition by both the TLRs

and cytosolic PRRs for IFN-α/β expression. TBK-1 is required for the phosphorylation

of the Ser 386 residue of IRF-3 that enables the transcription factor to dimerise and

to upregulate IFN-α/β transcription (Figure 11). Naturally viruses have evolved viral

evasion proteins to disable this critical signalling molecule.


22

Fig. 11. Viral interference of TBK-1. Rabies Virus P protein (Pi) and Hantavirus G1 protein interact with the TBK-1

complex inhibiting its function. Borna Disease Virus P protein (Pii) directly binds to the phosphorylation site of TBK-1

inhibiting it from phosphorylating IRF-3 (see text). Green arrows show inhibition of phosphorylation. Black dashed

arrows indicate simplified pathway. Modified from [70, 71].

Rabies Virus (RABV) and Hantaviruses are enveloped negative sense ssRNA

viruses. RABV causes haemorrhagic fever with virtually 100% mortality in non-

vaccinated individuals whereas Hantaviruses causes a less lethal haemorrhagic

fever with pulmonary and renal syndrome. It has been recently described that

activation of IRF-3 by TBK-1 is prevented by the RABV phosphoprotein P and the G1

cytoplasmic tail of Hantaviruses. Infection of cells containing TBK-1 expression

plasmid with P or G1 protein both led to a reduction of TBK-1 mediated IFN-α/β

transcriptional activity compared to controls of around 90% [71]. Currently the

mechanisms of action in blocking phosphorylation of IRF-3 by TBK-1 are not known.

The P protein however does not co-localise or immunoprecipitate with either IRF-3 or

TBK-1, suggesting an indirect interaction with TBK-1. In contrast the G1 protein

directly interacts with TBK-1 supported by immunoprecipitation experiments [70].


23

The nature of the G1 cytoplasmic tail determines the pathogenicity of Hantaviruses.

The G1 cytoplasmic tail of the pathogenic NY-1V, but not of the non-pathogenic

Prospect Hill virus (PHV) is able to suppress IFN-β expression. Deletion studies

showed that infection of cells with G1 negative NY-1V viruses stimulated IFN-β

expression and were cleared at similar levels to PHV. Non-pathogenic Hantaviruses

are not able to affect TBK-1 as they encode a different variant of the G1 cytoplasmic

tail (Figure 12) that exhibits 27% divergence from the pathogenic species [70].

Fig. 12. Amino acid alignment of 142-residue G1 tail sequences from NY-1V and PHV. There is high degree of

variation between the pathogenic NY-1V and the non-pathogenic PHV (see text) Residues which differ from NY-1V

are highlighted and bolded. Modified from [70].

The PHV G1 cytoplasmic tail was not able to immunoprecipitate with TBK-1 whereas

that of NY-1V interacts strongly with TBK-1. This indicates that differences within the

G1 tails which include charge changes, Proline insertions, Tyrosine insertions and

deletions, and the presence of an additional Cys 128 within the PHV G1 could

explain the differences in TBK-1 binding [70]. Non-pathogenic Hantaviruses are thus

unable to disrupt the Interferon Gateway, which causes their rapid clearance by the

innate immune response. This illustrates the causality between the potency of viral

immune evasion and pathogenicity, where the adaptation in the G1 cytoplasmic tail

allows pathogenic Hantaviruses to evade the Interferon Gateway early in infection

[72]. This leads to rapid viral replication and dissemination, causing the acute

pathology observed.

In contrast Borna Disease Virus, a zoonotic neurotropic virus, encodes a P protein

that binds strongly to the kinase domain of TBK-1 via a conserved Ser-X-X-X-Ser

recognition sequence [73]. In all cases the formation of IRF-3 dimers, nuclear import,

and transcriptional activity of IRF-3 is vastly reduced by the interference of TBK-1

kinase functionality by the P protein.


24

Targeting the IFN-α/β transcription Factors

Viruses may not be able to completely disrupt the TLR and the MDA5/RIG-I

signalling pathways, leading to activation of the transcription factors and IRF-3, IRF-7

that promote IFN-α/β expression. Viruses have thus evolved a multitude of strategies

to eliminate these transcription factors to prevent IFN-α/β expression and the

generation of the antiviral state (Figure 13).

IRF-3

P

Nucleus

IRF-3

P

IRF-3

P

CBP/p300

IRF-3

P

Proteasome

NSP1

Npro

ICP0

NSP1RTA

ML

vIRF-1E1A

E6

IRF-7

P

IRF-7

P

IFN-a

IRF-7

P

IRF-7

P

Degradation

IFN-bIRF-3

P IRF-3

P

CBP/p300

Fig. 13. Viral inhibition of IRF-3 and IRF-7. Rotavirus NSP1, BVDV/CSFV Npro, HSV ICP0 and HHV8 RTA viral

proteins target their respective IRFs to the proteasome for degradation (see text). CBP/p300 is a coactivator of IRF-3,

binding to IRF-3 and the IFN-β promoter. THOV ML, HHV8 vIRF-1 and Adenovirus E1A viral evasion proteins target

IRF-3 and CBP/p300 binding sites to prevent their interaction. E6 targets both IRF-3 and CBP/p300 (see text).

Modified from [74, 75].


25

Currently viral inhibitors of the essential transcription factors, NF-κB, ATF-2 and c-

Jun (see Introduction) have not been specifically identified in the context of IFN-β

expression. Transcriptional regulators of the NF-κB/IKKβ family promote the

expression of well over 100 target genes, the majority of which participate in the host

immune response [76]. Thus it is likely that viruses have evolved mechanisms to

evade this response, which need to be elucidated.

IRF-3/ IRF-7 degradation

Classical Swine Fever Virus (CSFV) is a Pestivirus sharing similar features to BVDV.

They both encode the non-structural Npro papaine-like cysteine protease [75, 77].

This protein is responsible for cleaving the C-terminus of the nascent polyprotein

generating the mature N-terminus of the nucleocapsid protein. Recent studies have

linked Npro with the proteasomal degradation of IRF-3 [75]. Deletion studies have

defined the requirements for IRF-3 degradation, demonstrating that the protease

function of Npro is not necessary for degradation, but that an intact, full-length, Npro is

absolutely required for its activity. This is supported by the observation that both N-

terminus and C-terminal truncation mutants lack the ability to degrade IRF-3.

The ubiquitin-proteasome degradation pathway is the main lysosomal-independent

protein degradation system that regulates the expression of cellular proteins involved

in metabolism [78]. IRF-3 is regulated by RO52, a cellular E3 ubiquitin ligase that

ubiquinates phosphorylated IRF-3 (as well as other key immune proteins) (Figure 14).

This is critical for the regulation of IFN-β signalling, where it is necessary to prevent

the production of IFN-β once a viral infection has been cleared, otherwise

immunopathology resulting from deregulated ISG expression could occur.


26

Fig. 14. The ubiquitin proteasome degradation pathway. Modification of eukaryotic proteins with ubiquitin (Ub)

prior to proteasome degradation requires an E1 activating enzyme, E2 conjugating enzyme, and an E3 ubiquitin

ligase. Ubiquitin is activated by E1 which is then transferred to E2. E3 covalently attaches ubiquitin to the Lys

residues of target proteins. Polyubiquitinated proteins are targeted to the proteasome and degraded in an ATP-

dependent manner. Taken from [78].

Npro subverts this pathway by interacting with IRF-3 and increasing the levels of IRF-3

ubiquitination, leading to proteasome mediated degradation. This was confirmed by

treating Npro expressing cells with proteasome inhibitor MG132, which returned levels

of IRF-3 to those of the control cells [79]. Neither BVDV nor CSFV Npro proteins co-

immunoprecipitate strongly with IRF-3, which suggests either a weak interaction or

that Npro requires a protein intermediate that contains or recruits a cellular protein with

E3 ubiquitin ligase activity. Recent studies demonstrate that BVDV Npro requires IRF-

3 to be polyubiquitinated as degradation only occurred at temperatures permissive

for E1 ubiquitin-activating enzyme activity [77]. This suggests that Npro may serve as

a platform to coordinate the premature ubiquitination of IRF-3, and consequent

proteasome mediated degradation.

BVDV exists as two pathological variants, cytopathic (cp) and non-cytopathic (ncp)

that can both cause acute infections. cpBVDVs are derived from ncp viruses by

mutations or genomic recombination. Only ncpBVDV is able to establish life-long

persistent infection following intra-uterine infection during the first trimester of

pregnancy [80]. The Npro protein of both cp and ncpBVDV specifically targets IRF-3

[77]. This indicates that the Npro protein degradation of IRF-3 is not the contributing

factor to the difference in pathology of the two variants of BVDV. Nevertheless,


27

N-Terminus C-Terminus N-Terminus C-Terminus

Generic C4HC3 domain Rotavirus NSP1 C4 domain

immunosuppression of the Interferon Gateway and ISGs could contribute to the

increased frequency and severity of secondary infections in BVDV-infected animals,

as IRF-3 is required for inducing IFN-β expression for bacterial infection and other

viral infections that are recognised by TLRs and cytosolic sensors [81].

Other viruses are able to target IRF-3 to the proteasome, but are the platform

themselves for recruiting the E1 and E2 ubiquitination proteins. Rotavirus NSP1 and

HSV ICP0 both contain the essential IRF-3 binding domain and RING finger that is

required for IRF-3 degradation, which is present on the cellular E3 ubiquitin ligase

RO52 [82-84]. RING fingers are Cys/His rich domains that facilitate the transfer of

ubiquitin from E2 to the substrate. These residues are spaced in a Cys 3 His Cys 4

(C3HC4) pattern that coordinates two zinc ions in a cross-brace motif (Figure 15).

This forms protrusions that act as docking sites for E2 and the protein substrate via

protein-protein interactions between the RING finger and the target protein [85].

Fig. 15. RING Finger Zinc (Zn) binding domains. The Structure of RING finger domains (see text). C = Cysteine;

H = Histidine. Modified from [83, 86].

HSV ICP0 shares significant sequence homology with the RO52 RING finger,

whereas the Rotavirus NSP1 RING finger is present in the opposite orientation in a

C4HC3 pattern compared to the conventional C3HC4 pattern observed in RO52 and

ICP0 (Figure 16). In light of this, and as both ICP0 and NSP1 RING fingers share

little sequence homology, this indicates that NSP1 and ICP0 independently evolved

this structural feature to mimic the functionality of RO52. The sequence similarity

between ICP0 and RO52 suggests that ICP0 acquired this feature via HSV genomic

DNA recombining with cellular DNA encoding RO52 or a similar ubiquitin ligase.


28

Fig. 16. Amino acid sequence alignment of NSP1, ICP0 and RO52 RING fingers. RO52 is the cellular E3

Ubiquitin ligase. HSV ICP0 and Rotavirus NSP1 share a RING finger domain. ICP0 shares significant homology with

RO52, whereas NSP1 has its RING finger in opposite orientation (see text). Bold red letters indicate cysteine and

histidine residues that bind to Zn2+. Modified from [87-89]

Mutational analysis of Cys and His residues within the conserved N-terminal zinc-

binding domain in NSP1 and ICP0 impaired their functionality. Sequence comparison

of Npro and Rotavirus NSP1 did not reveal any homology providing further evidence of

different modes of action [83]. Moreover, NSP1 is IRF-3 species specific, where

different strains of Rotavirus NSP1 demonstrate significant variation in their capacity

to induce the degradation of IRF-3 from other species [90]. This has implications in

vaccine design where instead of relying on the traditional method of animal and

human Rotavirus recombination to generate vaccine strains, mutation of the NSP1

gene of human Rotavirus strains may give rise to vaccine candidates that have

greater efficacy [91].

A recent study found that the HIV-1 accessory proteins Vpr and Vif, modulate the

antiviral response by targeting IRF-3 for degradation [92]. Mutational analysis

determined that Vpr and Vif worked in cooperation to induce the degradation of IRF-3,

although the exact mechanism is undefined. That HIV-1 has the capacity to decrease

cellular IRF-3 levels displays the need of HIV-1 to circumvent the innate antiviral

response during the early phase of viral replication.

Furthermore, NSP1 is also able to target IRF-7 to the proteasome for degradation via

the same mechanism as for IRF-3 [93]. The capacity of NSP1 to induce IRF-7

degradation may allow rotavirus to move across the gut barrier by enabling the virus

to replicate in dendritic cells and macrophages that constitutively express IRF-7 for

IFN-α production. The HHV8 Immediate-Early Transcription Factor RTA encodes E3

ubiquitin ligase activity like that of NSP1 and ICP0. Mutational analysis identified

three critical residues, Cys 131, Cys 141, and His 145 required for this function. RTA

targets IRF-7 for proteasome mediated degradation in pDCs thus preventing the

expression of IFN-α [94].

Rotavirus NSP1 43 C-L-D-C-C-...Q-H-T-D-L-T-Y-C-Q-G-C-L-I-Y-H-V-C-E-W-C-S-Q-Y-N-R-C-F-L-D 70

RO52 16 C-P-I- C-L-....D-P-F-V-E-P-V-S- I- E-P-V-S-I-E-C-G-H-S-F-C-O-E-C- I- S-O-V-G-K-G-G-G-S-V-C-P-V-C-R-O 55

HSV ICP0 8 C-P-I- C-L-E-D-P-S-N-Y-S-M-A-L-P-.................C -L-H-A-F-C-Y-V-C- I- T-R-W-I- R-Q-N- P-T-....C-P-L-C-K-V 49

javascript:if(window.name=='')%20%7b%7b%20window.location.href='./nil';%20%7d%7d%20else%20%7b%7b%20open_HOME('/UniPub/iHOP/go?ID1=1521715');%20%7d%7d%20


29

Ultimately the post-transcriptional down-regulation of IRF-3 and IRF-7 effectively

inhibits IRF-3/7 dependent antiviral gene expression initiated through either the TLR3

or RIG-I pathogen pattern recognition receptors.

Viral Disruption of the IRF-3/CBP/p300 complex

Viruses can also simply sequester IRF-3 from the IFN-β promoter. Thogoto Virus (THOV) is a

member of the Orthomyxoviridae family that infects animals and ticks. The THOV ML protein

is localised to the nucleus and interacts with specific domains in activated IRF-3 preventing it

from forming the transcription complex with CREB Binding Protein (CBP) and p300 [95].

CBP/p300 is a coactivator of IRF-3, binding to both IRF-3 and the IFN-β promoter. Mutational

studies determined that ML did not affect the phosphorylation and DNA binding domains of

IRF-3, but was instead found to disrupt the IRF-association domain. The IRF-association

domain is essential for homodimer formation and for the interaction with CBP/p300.

Expression of ML prevents the association of the IRF-3/CBP/p300 complex on the IFN-β

promoter. Interestingly, studies have failed to precipitate ML with IRF-3. Again, this suggests

either a transient interaction, or the recruitment of an intermediate protein, which is yet to be

identified. The disruption of IRF-3 function by ML may help to explain THOV persistence in

the tick reservoir, where no vertical or horizontal transmission takes place. Instead, THOV

can infect ticks without being eliminated, and when the ticks feed on their animal host the

virus is transmitted to a new host

In comparison to the ML protein, human Herpes Virus 8 (HHV8) and Adenovirus have both

evolved to target the CBP component of the IRF-3/CBP/p300 complex, again preventing it

from functioning. They are both dsDNA viruses that replicate in the nucleus, and are both

associated with oncogenesis [96]. HHV8 is a human tumour-inducing Herpesvirus, strongly

associated with Kaposi’s sarcoma cancers in AIDS patients. It has developed a unique

mechanism for antagonising IRF-3 and IRF-7 (as well as additional IRFs involved in other

aspects of immunity) by incorporating viral homologues of the cellular IRFs, called vIRFs [97].

HHV8 and Adenovirus encode the orthologue vIRF-1 and E1A proteins respectively that are

able to interact with the same regions on CBP and p300, cysteine/histidine element 3 (C/H3)

and Kinase Inducible X (KIX) domain (Figure 17) [98].


30

2067–21121454-1805HAT IBiD

vIRF-1

E1A

vIRF-1

E1A

E6

CBP Domain Structure

Fig. 17. CBP inhibition by Viruses. HHV8 vIRF-1 and Adenovirus E1A are able to bind to KIX and HAT domains.

All viral proteins including HPV E6 (see text) are able to bind to cysteine/histidine element 3 (CH/3 domains) which

contains the HAT region and specific sequence which is homologous to p300. The viral proteins indirectly bind to the

IRF-3 binding domain (IBiD). The KIX domain interacts with other transcription factors. The text box displays the

sequence homology between CBP and p300 and E6 binding residues. Modified from [97, 99, 100].

As mentioned previously, the numerous protein-protein interaction domains in

CBP/p300 are capable of recognizing multiple transcription factors such as IRF-3.

Activation of IFN-β depends on the formation of a nucleoprotein complex between

CBP/p300 and IRF-3. The coactivator function of CBP/p300 depends on its intrinsic

acetyl transferase activity located in the “Half a tetratricopeptide” (HAT) region

present within the CH/3 domain of CBP. Acetylation of histones leads to chromatin

relaxation while lysine acetylation can create docking sites favouring protein–protein

interactions. The KIX domain interacts with other transcription factors necessary for

IFN-β expression such as c-Jun. Thus by targeting these domains they are

preventing the formation of the necessary transcription factor complexes required for

IFN-β expression.

Human Papilloma Viruses (HPVs) go one step further by targeting both CBP and

IRF-3 via the E6 oncoprotein [100]. HPVs are small dsDNA viruses which infect

cutaneous and mucosal epithelia and can result in cervical cancer after viral genome

integration into host DNA. HPV16 E6 is able to bind to an E-L-L-G motif located in

the linker region of IRF-3. The specificity of E6 for CBP/p300 has been narrowed

down to a particular sequence at the C/H3 domain (Figure 17) that if deleted

prevents E6 binding [99]. However, the E6/IRF-3/CBP/p300 complex formed is

unable to bind to the IFN-β promoter region, thus preventing induction of IFN-β

expression.

Only certain high risk subtypes of HPV, such as HPV16, are associated with cervical

cancer. Functional studies have linked the level of pathogenicity to differences in the


31

E6 protein. Interestingly, E6 protein encoded by the low risk HPV6 subtype was

unable to bind to CBP and IRF-3, resulting in greater IFN-α/β production and the

generation of antiviral effecters [101].

IRF-7 is crucial for promoting IFN-α induction, but compared to IRF-3 less research

and fewer viruses have been identified for it. The HHV8 IRF-3 homologue vIRF-1

also blocks cellular IRF7-mediated innate immunity in response to viral infection [97].

HHV8 vIRF-3 specifically interacts with both the DNA binding domain and the central

IRF-association domain of IRF-7 via 40 amino acids containing double α-helix motifs.

This interaction leads to the inhibition of IRF-7 DNA binding activity and, therefore,

suppression of IFN-α expression and consequently IFN-α mediated immunity.

Other viruses that have been shown to sequester to IRF-3 and IRF-7 from their

respective promoters via undefined mechanisms include HHV6 IE1 and Ebola Virus

BZLF-1 respectively [102]. IE1 binds to phosphorylated as well as unphosphorylated

IRF-3 blocking IFN-β transcription. BZLF-1 is localised to the nucleus targeting

activated IRF-7 and possibly destabilising the homodimer [103]. The ssRNA Severe

Acute Respiratory Syndrome Coronavirus (SARS-CoV) is highly pathogenic in

humans, with 10% human mortality in the 2003 SARS outbreak [104]. The high

pathogenicity displayed suggests that SARS-CoV has developed mechanisms to

evade activation of the Interferon Gateway or to hyperactivate it, e.g. in the case of

1918 Influenza A pandemic where the majority of people died from immunopathology

rather than the virus itself [105]. SARS-CoV open reading frame (ORF) 3b, ORF6

gene products and N proteins bind to IRF-3 disrupting the IRF-3 homodimers [106].

All of the proteins are potent inhibitors of IFN-β which result in severe

immunodeficiency.


32

3. Viral interference of the JAK/STAT Pathway

IFN-α/β mediated effects rely exclusively on the JAK/STAT pathway for expression of

ISGs (see Introduction). Due to the pivotal role of this pathway in the immune

response, viruses have evolved mechanisms to inhibit multiple signalling levels of

this pathway obstructing the generation of the antiviral state in the infected cell.

3.1. IFNAR receptor disruption

VACV and most Orthopoxviruses encode soluble IFN-α/β binding proteins that are

homologues of the IFN-α/β receptor IFNAR. The VACV homologue B18R protein

displays broad species specificity for human, rabbit, cow, rat, and mouse IFN-α/β

[107], as it contains the highly conserved IFNAR IFN-α/β binding consensus

sequence PV---YV---KW---W---F---IFWENTS---VYCV [108]. Binding studies

indicated that B18R is expressed at far greater levels than IFNAR and binds with

greater affinity to IFN-α/β, out-competing IFNAR on two levels for IFN-α/β (Figure 18),

where IFNAR kinase auto-phosphorylation and STAT-1 phosphorylation was virtually

blocked by B18R.

Fig. 18. VACV inhibition of the JAK/STAT pathway. VACV B18R binds to IFN-α/β, sequestering it from the IFNAR

(see text) and inhibiting initiation of JAK/STAT signal transduction pathway (see text). Modified from [109].


33

Furthermore, the B18R gene is expressed as a 52kDa integral membrane protein

and a 65kDa secreted protein. The latter contains a signal peptide at the N-terminus

and lacks the C-terminal hydrophobic transmembrane domain of the 52kDa that

enables the 65kDa to be secreted [109]. The B18R protein is able to both prevent

the virus infected cell responding to infection and prevent the generation of antiviral

resistance in neighbouring uninfected cells.

3.2. Viral inhibition of JAK kinase Activity

Flaviviruses, members of the Flaviviridae family, show great diversity in their viral

evasion proteins. Japanese Encephalitis Virus (JEV) and Langat Virus (LGTV) both

encode the NS5 protein [110, 111]. JEV NS5 expression studies demonstrated

reduced levels of Tyk2 kinase autophosphorylation. This reduction in kinase activity

results in a significant depletion in the levels of STAT-1 phosphorylation in cells

expressing JEV NS5. Furthermore, LGTV has a wider ranging effect and is able to

also reduce the activity of Jak1 [110]. Mammalian two-hybrid system studies and co-

immunoprecipitation experiments revealed that JEV and LGTV NS5 did not interact

directly with the JAK kinases. In an attempt to identify the mechanisms of NS5

activity, cells were treated with a broad-spectrum inhibitor, Protein Tyrosine

Phosphatase (PTP). This suppressed NS5 IFN-α/β antagonism suggesting that NS5

acts as a platform for the recruitment of cellular PTPs which negatively regulate the

IFNAR JAK kinases, preventing downstream signalling (Figure 19).


34

Fig. 19. Viral interference with JAK kinase regulation. A) During the normal regulation of JAK/STAT pathway,

PTPs dephosphorylate the JAK kinases and STAT proteins inactivating them in the absence of IFN-α/β. The SOCS

proteins inhibit JAK kinase activity. B) In presence of IFN-α/β, these negative regulators dissociate upon IFNAR

dimerisation, where the JAK kinases phosphorylate and activate the STAT proteins. C) Viruses can inhibit the

phosphorylation of the STAT proteins (See text). Flavivirus NS4B and NS5 proteins can recruit PTP to deactivate the

JAK kinases. HPV E6 directly binds to Tyk2 inhibiting its kinase activity. HCV and HSV-1 upregulate SOCS3

expression, which negatively regulates Jak1 kinase activity. Green dashed arrows indicate phosphorylation. Blue

dashed arrows show dephosphorylation. Red arrows indicate viral mediated effects. Black dashed arrows show

localisation of proteins [112-114].

Other Flaviviruses such as Dengue Fever Virus (DFV), West Nile Virus (WNV) and

Yellow Fever Virus (YFV) do not inhibit JAK/STAT signalling via the NS5 protein

Instead, they encode the NS4B protein that has evolved the same function in

targeting STAT phosphorylation [112]. In NS4B recombinant plasmid infected cells,

STAT-1 proteins remained in the cytosol in an unphosphorylated state. However, the

WNV NS4B protein was found to specifically inhibit the activity of the JAK kinases,

preventing their autophosphorylation activities in the same way as LGTV and JEV

NS5 [115]. As the NS4B protein of these viruses share moderate sequence


35

homology (36%), it is possible that YFV and DFV subtypes share the same

mechanism although this has yet to be defined [112]. Other NS proteins have been

implicated in JAK/STAT interference, such as NS2A, NS4A for DFV subtypes, and

NS2B, NS3 for WNV and JEV. However, the exact role of these proteins is unclear

[116].

HCV, a Hepacivirus of the family Flaviviridae, encodes an NS4B protein that has little

sequence homology to those discussed. It employs along with the DNA virus HSV-1

the same strategy to inhibit JAK kinase mediated signalling. Firstly, through

mutagenesis studies HSV-1 and the core protein of HCV has been shown to

suppress IFN-α/β induced phosphorylation of Jak1, Tyk2, STAT-1, and STAT-2 by

upregulating the transcription of Suppressors of Cytokine Signalling (SOCS) 3 [113,

117]. SOCS3 is a cellular inhibitor that binds to the cytosolic domain of the IFNAR,

preventing the STAT-1 proteins from associating with the kinases (Figure 18).

Binding of IFN-α/β to IFNAR2 causes SOCS3 to become rapidly phosphorylated at

residues Tyr 204 and Tyr 221, by JAKs and other receptor tyrosine kinases [114].

However, the increase in SOCS3 cellular expression by HSV-1 and HCV overwhelms

this effect, resulting in inhibition of STAT-1 phosphorylation [113, 117]. HSV-1 also

encodes the ICP27 protein, which reduces the phosphorylation of STAT-1 in early

infection, although its precise mechanism of action is yet to be elucidated [118].

Overall downstream signalling is disrupted and the virus overwhelms the cell.

In contrast, co-immunoprecipitation studies revealed that the HPV E6 protein directly

interacts with the Tyk2 JAK Homology (JH) 6 and 7 (JH6-JH7) IFNAR binding

domains, restricting Tyk2 from binding to IFNAR1 and thus inhibiting its functionality

[119]. This was supported by genetic analysis of Tyk2, which identified the presence

of the E6 protein E-S-L-G binding sequence within the JH6 domain. As a result Tyk2

is unable to associate with IFNAR and thus phosphorylate the STAT proteins.

Furthermore, the benign HPV11 subtype E6 protein displayed decreased inhibitory

activity compared to HPV16 and 18 E6 proteins, further illustrating the importance of

the nature of the E6 protein in determining HPV pathogenesis.

The V and C proteins of MeV are able to inhibit JAK kinase signalling by interacting

with the scaffolding protein RACK1 (Receptor for Activated Kinase 1). RACK1 is a

member of the G protein family and acts as an adaptor protein between the IFNAR

and inactive (unphosphorylated) STAT-1 (Figure 20).

http://en.wikipedia.org/wiki/SOCS


36

Fig. 20. MeV inhibition of JAK kinase activity. MeV V and C proteins form a complex with IFNAR and the

associated scaffold protein RACK1. V and C complex formation prevents the STAT proteins from accessing the JAK

kinase phosphorylation domains, resulting in blockage of ISG expression (see text). Green dashed line indicates

phosphorylation. Red dashed line indicates viral evasion protein mediated recruitment of cellular proteins. Modified

from [120]

Molecular binding studies demonstrated that both viral proteins can interact with

RACK1. However, the V and C proteins vary in their specificity to the IFNARs. The

V protein interacts with IFNAR2 whilst the C protein interacts with IFNAR1, thereby

inhibiting the phosphorylation of the STAT proteins [120]. Co-localisation studies

revealed that the V protein additionally captures STAT-1, preventing it from being

phosphorylated by other IFNARs not bound to the V and C proteins. Other viral

proteins such as HIV nef protein and Adenovirus E1A have been shown to bind to

RACK1, but there have been no studies to date demonstrating whether they display

similar properties in the context of JAK/STAT inhibition.

3.3. STAT Protein Sequestration

Viruses are able to encode viral evasion proteins that act as STAT protein binding

platforms. They can sequester inactive STAT proteins from phosphorylation by the

JAK kinases and active STAT heterodimers from translocating to the nucleus in a

degradation independent manner (Figure 21), thus preventing ISG expression.


37

Fig. 21. Viral STAT protein sequestration strategies. The MeV SeV and HCV viral evasion proteins bind to

STAT1 preventing its phosphorylation. Nipah, Hendra V and RABV P proteins bind to both STAT-1 and STAT-2,

obstructing their dimerisation. SeV and HCV viral evasion proteins bind to phosphorylated STAT-1, preventing its

interaction with SH2 domain on STAT-2 (see text). Modified from [121-123].

The MeV P protein is able to bind to STAT-1 and prevent its phosphorylation by the

JAK kinases by binding to the Src Homology 2 (SH2) domain of STAT-1 (Figure 22)

[121].

Fig. 22. The STAT-1 structural domain. The SH2 domain is required for interacting with the JAK kinases and upon

phosphorylation at specific residues (see text) for binding to STAT-2 and IRF-9. Taken from [114].

The SH2 domain is a signalling modulating platform that enables the STAT proteins

to interact with each other, the JAK kinases and IRF-9. The JAK kinases

phosphorylate STAT-1 SH2 at Tyr 110, enabling it to mediate the formation of the

active STAT heterodimer by binding to the corresponding phosphorylated SH2

domain of STAT-2. Mutagenesis and co-immunoprecipitation studies showed that

the MeV P protein contains a Y-(Y/H)-V-Y-D-H sequence that is critical for the

disruption of STAT-1 phosphorylation. This is highly conserved within the P proteins


38

in the Morbillivirus genus, so presumably other P proteins of Morbilliviruses may

share these inhibitory properties.

Nipah Virus and Hendra Virus are from the Paramyxoviridae family. They are

emerging zoonotic pathogens harboured in fruit-bats [124]. The Nipah and Hendra

Virus V proteins and the RABV P protein are able to form a trimeric complex with the

STAT heterodimer, preventing its translocation to the nucleus. Mutagenesis studies

of V protein interactions showed that the V protein interacts with the SH2 binding

domain of phosphorylated STAT-1 [125, 126]. The V protein disrupts this interaction

by binding to SH2 and recruiting STAT-2 to a different part of STAT-1. In contrast

yeast two hybrid screening revealed that the RABV P protein interacts with both

STAT proteins in the active heterodimer by binding to their DNA binding and coiled

coil domains [123], forming an inactive complex and resulting in its retention in the

cytosol [127].

SeV and HCV both encode viral proteins that to bind to the STAT-1 SH2 domains,

conferring the dual advantage of preventing the interaction with the JAK kinases and

with STAT-2 [128]. The SeV C protein performs both of these functions whereas the

HCV NS5A binds to unphosphorylated STAT-1 and the core protein binds to

phosphorylated STAT-1. However, the SeV C protein only inhibits the

phosphorylation of Ser 727 but not Tyr 701 of STAT-1 [128]. These partially

phosphorylated, non-functional STAT-1 proteins are still able to bind to STAT-2, but

the heterodimer formed is unable to translocate to the nucleus. As viral evasion

proteins bind to their targets in competition with the normal cellular substrates, by

targeting both unphosphorylated and phosphorylated STAT-1, these viruses can

potently shutdown STAT-1 mediated signalling at two levels, thereby more efficiently

inhibiting ISG expression.

Additionally HCV and Hepatitis B Virus (HBV) are able to inhibit STAT protein

functionality by disrupting the dissociation of activated STAT-1 and STAT-2 from their

inhibitor, Protein Inhibitor of Activated STAT 1 (PIAS1). PIAS1 blocks the DNA-

binding activity of STAT heterodimers to ISG promoters to prevent the

overexpression of the ISGs which is harmful to the cell. When STAT proteins are

phosphorylated, they are methylated on Arg 31 by Protein Arginine Methyltransferase

1 (PRMT1), which allows them to dissociate from PIAS1. HCV and HBV both

upregulate the transcription of the PRMT1 inhibitor, Phosphatase 2A, thus preventing

PRMT1 from methylating the activated STAT proteins [129, 130]. As a result PIAS1


39

remains bound to the STAT protein DNA binding domain where the heterodimer is

unable to induce ISG expression.

3.4. Viral Induction of STAT Protein Degradation

Paramyxoviruses are able to target the STAT proteins for degradation via the

ubiquitin proteasome pathway (Figure 14). RSV NSP1 is able to act as an active

targeting platform, containing an E3 ubiquitin ligase domain and specific motifs that

recruit the E1 and E2 ubiquitin transferase proteins. The V proteins of certain

Paramyxoviruses share this function by encoding a highly conserved RING domain in

the C-terminus required for E3 ligase activity [131]. The V proteins and RSV NSP1

are able to interact with one or both of the STAT proteins to recruit them to the

platform for ubiquitination, thereby targeting the STAT proteins for proteasomal

degradation (Figure 23).

Fig. 23. Model of STAT protein degradation by viruses. Viral evasion proteins bind to the STAT proteins and

target them for proteasomal degradation by their intrinsic E3 ligase activity and recruitment of the ubiquitination

cofactors E1, E2, DDB1 and Cull4A (see text). Modified from [122].

RNA interference studies have determined that the STAT-targeting machinery

consists of additional cellular proteins including DDB1, an ultraviolet-damage induced

DNA binding protein and several members of the Cullin family of SCF ubiquitin ligase

subunits, including Cullin 4A. The RSV NSP1 and Paramyxovirus V proteins are

essentially subverting a normal cellular pathway by a combination of virus encoded

and host derived factors. In humans, the V protein of PIV5 and Mumps Viruses

target STAT-1 for degradation [122], whilst the PIV2 V protein, RSV NSP1 and the

SeV C protein induce the degradation of STAT-2 [67, 132, 133]. Proteasome

inhibition studies with MG132 showed that only the larger C and C’ variants of the

SeV C protein (Figure 9) were able to induce STAT-2 degradation, whereas the


40

smaller Y1 and Y2 forms were able to bind to STAT-1 but had little effect on cellular

STAT-1 levels [67].

In addition to sequestering STAT-1 from STAT-2, proteasome inhibition studies

revealed that the core protein of HCV induces the degradation of STAT-1 [134]. The

core protein does not encode E3 ubiquitin ligase activity, but acts through the

recruitment of an as yet undefined E3 ubiquitin ligase, possibly E6-AP which binds to

the core protein involved in the degradation of Retinoblastoma Tumour Suppressor

Protein implicated in HCV oncogenesis [135]. This illustrates the multifunctionality of

the HCV core protein in targeting the JAK/STAT pathway. The catalytic turnover of

STAT-1 allows Paramyxoviruses and HCV to dismantle the IFN-α/β induced antiviral

state of cells thus facilitating subsequent viral replication.

3.5. Viral Inhibition of STAT trafficking

Viruses can block the nuclear translocation of activated STAT heterodimers by

interfering with nuclear import factors. Nuclear import factors are proteins that

function as key gatekeepers to regulate the transport of the STAT protein

heterodimer from the cytosol to the nucleus of cells to induce expression of the ISGs

[136]. Ebola VP24 impairs the nuclear accumulation of the Tyrosine-phosphorylated

STAT heterodimer by specifically interacting with Karyopherin alpha 1 (Kα1), the

nuclear localisation signal receptor of STAT-1 (Figure 24). VP24 acts as a

competitive inhibitor, where overexpression of VP24 results in a loss of the

Kα1/STAT-1 interaction contributing to the block in IFN-α/β signalling [137]. This

results in an inhibition of IFN-α/β induced gene expression and the generation of the

antiviral state.


41

Ka1

VP35

Nucleus

IRF-9

Nuclear Translocation Complex

P STAT-2

PSTAT-1

P STAT-2

PSTAT-1

P STAT-2

PSTAT-1

P STAT-2

PSTAT-1

Ka1

Fig. 24. Viral disruption of ISGF3 complex. The STAT heterodimer is recognised by Karyopherin alpha 1 (Kα1),

for import into the nucleus which then forms a nuclear translocation complex with Kα2 and Kβ1. Ebola Virus VP24

acts as a competitive inhibitor of Kα1, sequestering the STAT heterodimer. SARS-CoV ORF6 protein is bound to the

Rough Endoplasmic Reticulum (RER) and Golgi body membranes, interacts with Kα2, which in turn recruits Kβ1. The

depletion of free Kβ1 and Kα2 in the cytosol blocks formation of the nuclear translocation complex, thereby

preventing the import of the STAT heterodimer into the nucleus. Modified from [137].

SARS-CoV ORF6 protein also prevents STAT heterodimer translocation. However,

this protein sequesters the nuclear import factors in a different cellular compartment

[138]. Uniquely among IFN-α/β antagonists which are localised in the cytosol, the

ORF6 protein is localised to the Rough Endoplasmic Reticulum (RER) and Golgi

membrane in infected cells, whereby it cannot be imported into the nucleus. The

ORF6 protein disrupts the nuclear translocation complex formation by indirectly

sequestering the essential nuclear import factor Karyopherin β 1(Kβ1). It binds to the

Kβ1 associate import protein Karyopherin alpha 2 (Kα2), tethering Kα2 to the

RER/Golgi membrane but leaving the N-terminus Kβ1 binding domain exposed. The

subsequent recruitment of Kβ1 to the RER/Golgi membrane depletes Kβ1 in the

cytosol, leading to a loss of STAT heterodimer translocation into the nucleus in

response to IFN-α/β signalling.

Unlike the other Paramyxoviruses described, the V protein of MeV does not degrade

the STAT proteins, where confocal microscopy revealed that it instead blocks nuclear

translocation of the STAT heterodimer which accumulated in the cytosol of virus

infected cells [139]. This observation is supported by affinity chromatography which


42

demonstrated that the V protein co-purified with phosphorylated STAT-1 and STAT-2.

Furthermore, MeV infection has been shown to relocalise STAT proteins to cytosolic

bodies. This indicates that MeV prevents the STAT heterodimer from interacting with

the appropriate nuclear import factors for nuclear translocation and thus the

expression of ISGs, although the mechanism remains to be elucidated.

3.6. ISGF3 Promoter Interference

Following nuclear translocation of the STAT heterodimer, the formation of the ISGF3

complex in the nucleus is critical for DNA binding of transcription factors and ISG

expression. Viruses have developed specific strategies for abolishing ISGF3

interaction with the ISG promoter.

Human Cytomegalovirus HCMV IE1 and HPV E7 proteins disrupt the formation of

ISGF3 by binding to the STAT heterodimer and IRF-9 respectively [140, 141]. Co-

localisation and immunoprecipitation studies demonstrated that HPV E7 directly

binds to IRF-9 in the nucleus, whilst HCMV IE1 interacts with the STAT heterodimer

but not IRF-9. In contrast, the RABV P protein counters ISGF3 promoter binding by

interacting with STAT-1 and interfering with its DNA binding activity. The Adenovirus

E1A protein disrupts transcriptional responses by sequestering the transcriptional co-

activator CBP/p300 required for activation of the ISG promoter. CBP/p300 interacts

with ISGF3 in a similar mechanism as for IRF-3 degradation (Figure 13).


43

4. Viral Interference of ISGs

Viral inhibition of IFN-α/β expression and signalling takes time to establish and is

never fully effective. Therefore, to replicate successfully, viruses also have to evade

a multitude of ISG products that are present initially at low levels within most cells.

4.1. PKR

The importance of PKR to the antiviral response is highlighted by the observation

that viruses have evolved mechanisms to inhibit all aspects of PKR function (Figure

25).

PKR

PKR

PKR

dsRNA

P

P

eIF2a

P

eIF2a

Inactive forInitiation of Translation

Active forInitiation of Translation

ActiveKinase

InactiveKinase

Viral dsRNA homologuesAdenovirus E1AEBV EB1HCV IRES

Mediators of eIF2aDephosphorylationHPV E6HSV ICP34.5

PKR Binding ProteinsHCV E2HCV NS5AHIV TatHSV Us11Influenza A NS1VACV E3LVACV K3L

dsRNA binding ProteinsEbola Virus VP35EBV EB2HSV Us11Influenza A NS1Rotavirus Sigma3VACV E3L

Fig. 25. Virus interference of PKR. Viruses are able to inhibit all stages of PKR activation and functionality (see

text). Green dashed arrows indicate phosphorylation. Modified from [4, 8, 142, 143].

In order for PKR to be activated it must first bind to viral dsRNA. As mentioned

previously, the VACV E3L, Influenza A NS1, Ebola Virus VP35, Rotavirus Sigma3,

HSV Us11 and the EBV EB2 proteins are able to bind to viral dsRNA and sequester

it from the TLRs and cytosolic sensors. These viral proteins, as expected, have the

additional effect of sequestering viral dsRNA from PKR. As a result, PKR remains

inactive in the cell leading to continued viral protein translation and viral replication.

The HSV Us11 dsRNA-binding domain is also able to bind to PKR in a dsRNA

independent manner to prevent PKR mediated apoptosis of the cell. The necessity

of this interaction evolved as the lytic cycle places the cell under stress, which

activates cellular factors, including the PKR-activating protein (PACT) [144]. The


44

critical nature of Us11 is revealed in Us11 knockout viruses, where PACT correctly

binds to PKR and initiates the apoptotic cascade due, in part, to translational

repression (Figure 26). Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick

End Labeling and co-localisation studies showed that PACT still binds to PKR but the

interaction of Us11 with the RNA binding domain disrupts the formation of the correct

PKR conformation by PACT, thus preventing the initiation of apoptosis cascades.

Fig. 26. HSV interference of dsRNA independent PKR activation. A) In the absence of cellular viral stress signal,

PKR is inactive where the kinase domain interacts with dsRNA binding domain 2 (dsRBMII) preventing PACT from

interacting with PKR. B) Upon binding of cellular stress signal, the kinase domain changes conformation allowing

PACT to bind to PKR, activating the kinase. C) Us11 C-terminus interacts with dsRBMI and dsRMBII, preventing

PKR from changing fully conformation and being activated by PACT. Modified from [144].

Mutagenesis studies determined that the R-X-X-X-P motif of Us11 is critical for

binding to PKR [144]. As the EBV EB2 protein contains this motif, it is possible that it

shares the inhibitory properties of Us11.

PKR domain interaction

Several viruses have evolved specialized mechanisms designed to inhibit the activity

of PKR by expressing dsRNA homologues that bind directly to PKR, (Figure 27).

Adenovirus encodes the virus-associated RNAs I and II (VAI and VAII) that are

required for efficient translation of viral and cellular mRNAs late in infection [145].

The HCV Internal Ribosome Entry Site (IRES) is not a separately produced viral

dsRNA homologue, but is an integral component of the HCV viral genome. The HCV


45

IRES is involved in recruiting the host cellular 40S ribosome subunit and other

cellular factors that are essential for the genomic translation of HCV. The HCV IRES,

Adenovirus VAI, VAII and EBV EB1 contain extensive RNA secondary structures that

interact with the PKR kinase domain impeding PKRs phosphorylation activity (Figure

27).

EBV EB1 HCV IRESAdenovirus VAI

5'

3'

5' 3'

5' 3'

Fig. 27. The viral dsRNA homologues. The Adenovirus VAI, EBV EB1 and HCV IRES viral products exhibit

significant secondary structures where specific STEM loops are critical for PKR inhibition (see text). Modified from

[145-147].

These homologues contain structural features that enable them to outcompete viral

dsRNA for interacting with the PKR dsRNA binding region. The homologues do not

have a 5’ cap or 3’ polyadenylated tail, but instead end in stretches of oligo-(U) that

optimizes efficiency of binding. The double-stranded stem and stem-loop regions of

STEM IV for VAI and EB1 and STEM IIIb for the HCV IRES contain a highly

conserved region that is involved in binding to the PKR dsRBMI domain [145-147].

Protein binding studies coupled with mutagenesis studies have determined that these

highly conserved regions contain a common central domain of GGGU and ACCC

that is critical for binding specifically to the dsRBMI domain (Figure 28). The PKR

kinase domain remains in an inactive state bound to dsRMBII, thereby allowing the

continuation of cellular and viral protein synthesis.


46

Fig. 28. Viral interference of the PKR domains. Viral evasion proteins bind to specific domains of PKR (see text).

Modified from [145-147].

Following the binding of viral dsRNA to PKR, dimerisation is necessary for trans-

autophosphoryation which activates the eIF2α kinase domain [148]. HCV encodes

two proteins, NS5A and the E2 envelope glycoprotein that are able to block PKR

dimerisation. Certain strains of HCV contain the Interferon Sensitivity Determining

Region (ISDR) in NS5A that is essential for the interaction with PKR. The presence

of ISDR is a virulence determinant, where those HCV strains that do not contain this

region display significantly decreased pathology in infected individuals, with patients

more likely to be successfully treated with IFN-α [149]. Protein binding studies

identified that NS5A ISDR binds to the PKR autophosphorylation domain [150].

Unglycoslated E2 is localised in the cytosol, binding to the PKR eIF2α domain and

preventing its phosphorylation [151]. As a result PKR autophosphoylation is

prevented by NS5A and any activated PKR in the cytosol is immediately neutralized

by E2, preventing PKR mediated viral and cellular mRNA degradation.

Additionally, the HCV E2 protein can be post-translationally glycosylated in the RER,

where it is consequently localised to the RER, counteracting the effect of the ISG

PKR related PERK (PKR-like ER kinase) [152, 153]. PERK is activated upon RER

stress which can be caused by the lytic cycle of HCV. Activation of PERK results in

the phosphorylation of eIF2α, thereby inhibiting cellular and viral protein synthesis.

HCV E2 inhibits PERK leading to continued viral replication in the cell [152].

VACV encodes two proteins that bind to different domains of PKR, increasing the

inhibition of PKR compared to inhibiting one domain alone. The VACV E3L protein

binds to the eIF2α kinase domain whereas the K3L protein binds to the PKR

substrate recognition domain [154]. Mutagenesis studies revealed that E3L PKR

inhibitory activity is dependent upon the residues Lys 167 and Arg 168 contained

within the E3L C-terminal dsRNA binding domain. Furthermore, deletion of the N-

terminus of E3L reduced inhibition by 1000 fold, indicating that both the E3L N-

terminus and C-terminus domains are essential for inhibiting PKR [155]. Gene


47

sequencing studies determined that K3L shares 30% amino acid homology with the

N-terminus of eIF2α, where K3L contains the eIF2α PKR binding motif K-G-Y-I-D at

position 72–83 [156].

Recent studies have shown that HHV8 vIRF-2 and influenza A NS1 protein inhibit

PKR by mechanisms that remain to be elucidated. Protein binding studies

determined that vIRF-2 binds to the PKR dimerisation domain whilst NS1 binds to the

PKR regulatory domain [157] [158]. As many different viruses use an immense

variety of strategies to evade PKR, this illustrates the importance of PKR in innate

immunity against viruses.

In contrast to viruses that inhibit PKR, HIV-1 subverts PKR mediated responses to

enhance its own replication in the cell. HIV-1 encodes the Tat protein that is

phosphorylated by PKR, enhancing Tat binding efficacy to HIV TAR RNA. The

Tat/TAR interaction is crucial for HIV viral replication as it increases transcription of

HIV mRNA. The phosphorylation of Tat at residues Ser 62, Tyr 64, and Ser 68

increases viral replication 100 fold compared to unphosphorylated Tat/TAR

interactions [159]. Furthermore, phosphorylated Tat downregulates PKR kinase

activity by acting as a pseudo-substrate of eIF2α [160], although this mechanism

remains undefined.

PKR degradation

Immunoprecipitation studies coupled with pulse-chase experiments revealed that

Poliovirus is able to degrade PKR, although the mechanism remains poorly defined

[161]. Protein binding studies determined that the Poliovirus encoded proteases 2A,

3C, and 3CD are not involved in the degradation of PKR, suggesting that Poliovirus

recruits a cellular protease or targets PKR for proteasomal degradation by encoding

or recruiting an E3 ubiquitin ligase.

Viral Targeting of Phosphorylated eIF2α

Viruses can target phosphorylated eIF2α (PeIF2α) for dephosphorylation, inactivating

the PeIF2α mediated block of cellular and viral mRNA translation. In the early stages

of HSV viral infection HSV ICP34.5 protein performs this role by recruiting the cellular


48

phosphatase Protein Phosphatase 1 alpha (PP1α) that dephosphorylates PeIF2α

(Figure 29).

eIF2a eIF2a

Uninfected Cell Virus Infected Cell

eIF2a eIF2a

eIF2a eIF2a

ICP34.5 PP1a

GADD34 PP1a

eIF2a eIF2a

HSV Infected Cell

GADD34 PP1a

E6

HPV Infected Cell

PKR

PKR

P

P

PP

P P

A B

C D

Fig. 29. Viral interference of eIF2α regulation. A) In uninfected cells GADD34 recruits PP1α which

dephosphorylates PeIF2α to prevent a block in cellular protein synthesis which is detrimental to the cell. B) In virally

infected cells PKR phosphorylates eIF2α, inhibiting cellular and viral protein synthesis. C) HSV ICP34.5 protein

recruits PP1α which then dephosphorylates PeIF2α. D) HPV E6 recruits GADD34 and PP1α, dephosphorylating

PeIF2α. Green dashed lines show phosphorylation. Blue dashed lines show dephosphorylation. Modified from [162,

163].

HSV ICP34.5 shares homology with the cellular regulator of PP1α, GADD34. In

uninfected cells, GADD34 recruits PP1α to PeIF2α which is then dephosphorylated.

This maintains a pool of unphosphorylated eIF2α that participates in the initiation of

translation of cellular mRNAs. HSV ICP34.5 subverts this regulatory mechanism by

recruiting PP1α to PeIF2α [162]. This was demonstrated by cell infection studies

where eIF2α remained phosphorylated and cellular and viral protein synthesis

remained blocked in cells infected with HSV ICP34.5 negative mutants.

HPV18 has evolved a similar mechanism for modulating PKR activity, but instead of

encoding a homologue for GADD34, the HPV E6 protein acts as a platform for

recruiting GADD34 which in turn recruits PP1α to promote the dephosphorylation of

PeIF2α [163]. The mechanism utilized by E6 to promote this is currently unknown.

However, the nature of the E6 protein again influences HPV pathogenicity. The

oncogenic HPV18 E6 protein is able to colocalise both in the nucleus and the cytosol


49

as opposed to the benign HPV11 E6 protein which is predominantly localised to the

nucleus and is thus unable to significantly promote PeIF2α dephosphorylation. HPV

and HSV are essentially subverting the normal cellular regulatory mechanism of

PeIF2α to prevent it blocking cellular and viral protein synthesis.

4.2. RNase L

Many viruses block the 2'-5' oligoadenylate synthetase (2’-5’ OAS) RNase L

activation pathway (see introduction), either by expressing dsRNA-binding proteins

as mentioned previously, or by other mechanisms of action (Figure 30).

2'-5' OAS

RNase L

RNase L

dsRNA

RNase L

RNA Degradation

InactiveEnzyme

ActiveEnzyme

Viral 2'-OA HomologuesHSV

dsRNA binding ProteinsEbola Virus VP35EBV EB2HSV Us11Influenza A NS1Rotavirus Sigma3VACV E3L

ATP 2'-5' OA

2'-5' OAS InhibitorHCV NS5A RLI Increased expression of RLI

HIV

Fig. 30. Viral inhibition of RNase L. Upon viral infection 2’-5’ Oligoadenylate Synthetase (2’-5’ OAS) binds to viral

dsRNA. 2’-5’ OAS converts ATP to 2’-5’ linked oligoadenylates (2’-5’ OA) which bind to RNase L, causing: 1) its

dissociation from the RNase L Inhibitor (RLI) and 2) RNase L dimerisation. The active RNase L dimer degrades

cellular and viral dsRNA. Like PKR viral inhibition, viruses target all stages in RNase L activation (see text). Modified

from [164].

HSV-1 and 2 encode 2’-5’ oligoadenylate homologues that directly compete with

cellular 2’-5’ oligoadenylate for RNase L. These 2’-5’ OA derivatives are weak

activators of RNase L, resulting in a profound decrease in RNase L activity in HSV

infected cells [165].

HCV evades RNase L action via two different strategies. Firstly, RNase L mediated

cleavage of HCV mRNA selects RNase L resistant variants [166]. RNase L degrades

HCV mRNA by cleaving predominantly after UA and UU dinucleotides in single-

stranded regions. HCV mRNAs from relatively IFN-α resistant genotypes (HCV 1a

and 1b) have fewer UA and UU dinucleotides than those of IFN-α sensitive

genotypes (HCV 2a, 2b, 3a and 3b). Patients infected with HCV 1b viruses are cured


50

less frequently than patients infected with HCV genotype 2 or 3. During IFN-α

therapy, HCV 1b mRNA accumulates silent mutations preferentially at UA and UU

dinucleotides, evading RNase L activity, and perhaps explaining the differences in

pathogenicity between HCV genotypes.

Secondly the HCV NS5A protein competes with viral dsRNA, binding to the active

sites of 2’-5’ OAS [167]. Cell culture assays revealed that NS5A physically interacts

with 2’-5’ OAS, where mutagenesis studies elucidated that the NS5A N-terminus (a. a.

1–148) NS5A and two separate regions of 2’-5’ OAS (a. a. 52–104 and 184–275) are

necessary for this interaction. Virus rescue assays confirmed this observation, which

revealed that the NS5A C-terminal ISDR region and PKR binding domain were not

required, as cell infected with NS5A N-terminus successfully counteracted the activity

of RNase L to the same degree as cells infected with full length NS5A.

RNase L is inactive in cells infected with HIV-1, remaining bound to its inhibitor.

Time course infection studies showed that HIV-1 induced the expression of the

RNase L inhibitor (RLI) by an as yet undefined mechanism [168]. The RLI protein

contains an ATP binding cassette that forms a heterodimer with RNase L, inhibiting

the binding of 2’-5’ OA to RNase L in a non-competitive manner [169]. The

downregulation of RNase L activity by HIV-1 contributes to the inhibition of the innate

intracellular immune response and the inability of patients to clear HIV-1 infection.

4.3. APOBECs

APOBEC3G and the closely related APOCBEC3F are expressed mainly in T

lymphocytes and macrophages, which are the main targets of HIV. In cells

infected with HIV mutants negative for the Vif HIV viral protein, the

APOBECs are packaged into HIV virions during viral assembly. During HIV

reverse transcription, the APOBECs deaminate deoxycytidine residues to

deoxyuridine (dU) in the growing minus-strand viral DNA [170]. These

dU-rich transcripts are either degraded or yield G-to-A hypermutated

nonfunctional proviruses. Vif prevents the incorporation of the APOBECs into the

virion by recruiting a cellular ubiquitin ligase (Cul 5), which targets the antiviral

proteins for proteasomal degradation [171, 172].


51

4.4. ADAR-1

ADAR-1 is a member of the multigene family of RNA editing enzymes that catalyse

the Carbon-6 deamination of adenosine (A) to yield inosine (I) in double-stranded

RNA structures [173]. VACV E3L protein disrupts this by binding to ADAR-1 via the

E3L dsRNA binding domain, inhibiting ADAR-1 deaminase activity. As a result

VACV prevents A-to-I editing by ADAR-1 [174].

4.5. Tetherin

It has been recently reported that IFN-α/β induces the expression of a cellular

membrane protein called tetherin. Tetherin binds to the viral envelope, preventing

viral budding. However, HIV encodes the Vpu protein that is able to bind to tetherin,

inhibiting its activity by an undefined mechanism. A possible mechanism involves

Vpu having intrinsic ubiquitin ligase activity like the HHV8 K5 protein which reduces

cellular levels of tetherin by targeting it for proteasome mediated degradation [175].

It is highly likely that many enveloped viruses are affected by tetherin, and have

evolved mechanisms to evade this.

4.6. PML

Certain viruses induce the disruption of PML nuclear bodies by targeting PML for

proteasome mediated degradation. PML interacts and regulates p53, a cellular

transcription factor involved in initiating the apoptosis pathway in response to cellular

stress such as that associated with virus infection [176] . In HSV-1 infected cells,

ICP0 accumulates in PMLs resulting in the induction of PML degradation via ICP0s

RING domain which functions as an E3 ubiquitin ligase [82]. Similar PML disruptions

were observed in cells infected with HCMV, EBV, HPV and Adenoviruses [177, 178].

As many DNA viruses target PML, this suggests that they disassemble these

nuclear structures to prevent the induction of cellular apoptosis which would destroy

the virus, or that PMLs are perhaps needed for their replication, but is yet to be

proven [35].

RNA viruses can also interact with PML, and this has been specifically shown for

immune evasion for HCV and RABV. Cell transfection assays showed that the HCV


52

core protein co-localises with PML and p53 [179]. This indicates that the HCV core

protein may compromise the pro-apoptotic function of p53, contribution to the

formation of HCV induced hepatocellular carcinoma. The RABV P protein binds to

PML, subverting its localisation from the nucleus to the cytosol [180]. This

mechanism sequesters PML from the nucleus inhibiting its antiviral activity, where

RABV replication is enhanced compared to cells infected with RABV P negative

mutants.


53

5. Discussion

The Interferon Gateway is the lynchpin of the host defence against virus infection.

Without it, viruses would completely overwhelm the host before the adaptive immune

system had a chance to respond. The antiviral state, whilst it may not be able to

eliminate the majority of pathogenic virus infections, is able to curtail virus

dissemination through a variety of sophisticated mechanisms. This is best illustrated

in knockout mice which have key deletions in the IFNAR receptor, making them

unresponsive to IFN-α/β [181]. These mice quickly succumb to viral infections despite

having a normal adaptive immune system. This is observed in humans, where infants

succumb to viral infections if they inherit genetic defects in the Interferon Gateway

[182]. Clearly, viruses that had not evolved IFN-α/β evasion strategies would now be

extinct. Consequently we observe that both RNA and DNA viruses have developed

an impressive array of mechanisms to surmount all levels of the Interferon Gateway

(Figure 31).

Viral Nucleic Acids

TLR Pathway RIG-I/MDA5 Pathways

JAK/STAT Pathway

InterferonStimulated

Genes

RNA VirusesBVDVEbola VirusInfluenza ARotavirus

DNA VirusesEBVHSVVACV

Transcription FactorActivation Pathway

RNA VirusesHCV

DNA VirusesVACV

RNA VirusesHCVInfluenza AMeVMuVPIVRSVSeV

RNA VirusesBorna Disease VirusBVDVCSFVEbola VirusInfluenza ANY-1VRABVRSVRotavirusSARS-CoVTHOV

DNA VirusesAdenovirusHHV6HHV8HSV

RNA VirusesHCVHendra VirusJEVLGTVMeVMuVNipah VirusPIVRABVSARS-CoVSeVWNV

DNA VirusesHBVHCMVHPVHSVVACV

RNA VirusesHCVHIVRABV

DNA VirusesAdenovirusEBVHHV8HPVHSVVACV

Fig. 31. Viral Domination of the Interferon Gateway. Some viruses encode more than one strategy to counteract

the effects of IFN-α/β and are able to act at multiple levels (see text).


54

Viruses are able to inhibit the whole continuum of IFN-α/β mediated antiviral

responses by targeting multiple levels of the strategic components of IFN-α/β

production, signalling and ISG effecter pathways. Disrupting only a solitary

intermediary component of the Interferon Gateway may lead to detection of the virus

by another pathway, leading to inhibition of viral replication. Most of the viral evasion

proteins are competing with normal binding domains of cellular receptors, signalling

adaptor proteins or the ISGs for specific substrates. Leakage occurs where the

functionality of these proteins and their constituent pathways are still active. For

example, the V protein of PIV5 disrupts MDA5 recognition of dsRNA in infected cells,

but is unable to completely inhibit the MDA5 signal transduction pathway and also

prevent TLR recognition and down stream signalling [183]. However, the V protein is

able to target the degradation of STAT-1 in the JAK/STAT pathway, leading to an

eventual decay (24–48 hours) of the antiviral state, as the maintenance of this is

impossible without continuous IFN-α/β signalling thereby facilitating subsequent viral

replication [184].

Furthermore, viral evasion proteins that cover the whole spectrum of the IFN-α/β

response converge on key signalling mediators that in turn affect the expression and

functionality of multiple downstream signalling pathways and effecters. This is

exemplified by the extensive viral targeting of TBK-1 and IRF-3 which mediate the

expression of IFN-α/β from the MDA5, RIG-I and TLR pathways. Many viruses such

as JEV specialise in targeting the JAK/STAT pathway as this the only route for IFN-

α/β mediation of induction of ISG expression. A common critical viral evasion

mechanism is the ability of the viral evasion protein to recruit or have the intrinsic

function of a cellular E3 ubiquitin ligase to target cellular antiviral signalling

components for proteasomal degradation. Many viruses target dsRNA, as this has

the potent dual effect of preventing the activation of multiple signalling pathways,

subsequent IFN-α/β expression and the consequent generation of the antiviral state,

but also of inhibiting ISGs such as PKR and RNase L that are viral dsRNA dependent.

5.1. Nature of Viral Inhibition

Viral evasion proteins expressed either separately or in combination are not able to

completely disable the Interferon Gateway, suggesting three possibilities. Firstly,

viral evasion proteins do not have the intrinsic capability to completely inhibit the

Interferon Gateway, but are still undergoing evolution to optimise this function.

Secondly, the development of complete IFN-α/β inhibition by viral evasion proteins


55

could be interpreted as a cellular stress signal activating other cellular antiviral

responses. A complete shutdown of IFN-α/β signalling could thus activate apoptotic

cascades through unidentified regulator mechanisms, or activate other arms of the

immune system. The latter has been observed in relation to HCV infection. Heat

shock protein 70 protects cells against oxidative stress, and suppresses the activity

of PKR and other related kinases such as PERK as their activity is detrimental to the

uninfected cell. IFN-α induces expression of Hsp70, preventing apoptosis in hepatic

stellate cells by cytoxic T lymphocytes which secrete type II IFN gamma, a potent

inducer of the ISGs [185]. Thus it would be detrimental for HCV replication if HCV

viral evasion proteins orchestrated the complete inhibition of the Interferon Gateway,

where this concept could be true for other viruses. Thirdly, it may not be beneficial to

completely disrupt the Interferon Gateway as the virus could use this response as

means of regulating virus replication. Cell stress caused by an exorbitant high rate of

viral replication could induce cellular apoptosis, killing the cell before the virus has a

chance to assemble into a fully functioning virion.

5.2. Comparing RNA and DNA Viral Evasion Strategies

As discussed previously, viruses can inhibit multiple levels of the Interferon Gateway.

By focusing mainly on HCV, Influenza A, HSV and VACV, these offer prime case

studies of the similarities and differences between RNA and DNA virus viral evasion

strategy (Table 2). RNA viruses, despite having smaller genomes than DNA viruses,

are equally capable of inhibiting the Interferon Gateway. This is because both DNA

and RNA viral evasion proteins use conserved functions to target the gateway. This

is due in part to viruses facing a continuous downward selective pressure on genome

size. The more viral evasion proteins a virus encodes the more time and cellular

resources it takes for the virus to replicate [186].Thus viruses containing fewer genes

have a faster replication rate and can quickly outcompete virus species with a greater

number of genes. However, this is balanced by the selective pressures applied by

Interferon Gateway via the action of ISGs. Those viral subspecies that can encode

proteins that disrupt the action of cellular antivirals or IFN-α/β expression and

signalling pathways leading to ISG expression would have a survival advantage over

viral species that do not.


56

Table 2

Virus Nucleic

acid

Number of

genes

Protein Pathway Mechanism of Action

HCV +ve

sense

ssRNA

10 NS3/4A TLR3 signalling Cleaving TRIF

RIG-I/MDA5 Cleaving IPS-1

Core JAK/STAT Upregulating SOCS3 (Jak1

inhibition)

JAK/STAT STAT-1 degradation

NS5A JAK/STAT STAT-1 Binding

ISG 2’-5’ OAS binding

ISG PKR binding

IRES ISG PKR binding

E2 ISG PKR and PERK binding

Influenza A -ve

sense

ssRNA

11 NS1 TLR3 dsRNA binding

ISG PKR dsRNA binding

ISG PKR binding (dsRNA independent)

ISG RNase L, dsRNA binding

RIG-I RIG-I, IPS-1 binding

VACV dsDNA 250 E3L TLR3 signalling dsRNA binding

ISG PKR, dsRNA binding

A46 TLR3/4 signalling Binding to TIR

A52 TLR3/4 signalling Binding to TRAF6

B18R JAK/STAT Homologue of IFNAR

K3L ISG PKR, Homologue eIF2α

HSV dsDNA 74 Us11 TLR3 dsRNA binding

ISG PKR, dsRNA binding

ISP0 JAK/STAT IRF-3 degradation

ICP27 JAK/STAT Inhibits STAT-1 phosphorylation

ICP34.5 ISG Dephosphorylate eIF2α

2’-5’ OA

homologues

ISG RNase L negative regulators

Viral Case Studies (see text)

Overall, the above selective pressures favour viral evasion proteins to contain two

features; one is for viruses to encode a conserved mechanism whose mode of action

is able to target many pathways, as discussed previously. Secondly, there is a

selective pressure on individual proteins to contain as many of these conserved

functions as possible within a single protein to minimise the genetic material required.

Influenza A is a prime example, encoding the NS1 protein that inhibits IFN-α/β

signalling and ISG action by sequestering dsRNA and also by a dsRNA independent

mechanism. The NS3/4A serine protease of HCV is able to act on both the TRIF

adaptor protein of TLR3 and IPS-1 of RIG-I signalling. DNA viruses such as VACV

and HSV encode dsRNA binding proteins (E3L and Us11 respectively) that exhibit

the same function. Conservation of function is further observed where many viral

evasion proteins originally evolved from those that are essential for viral replication


57

and assembly. For example, the HCV E2 envelope glycoprotein is involved in

initiating virus attachment to the host cell as well as inhibition PKR and PERK.

DNA viruses can contain many more genes than RNA viruses, where VACV contains

250 genes compared to Influenza A with 11 [187, 188]. This allows DNA viruses to

incorporate a greater number of novel genes from the cell through recombination with

cellular DNA, pirating them into the viral genome. In contrast, RNA viruses less

successfully recombine with cellular mRNAs due to RNA viruses’ limited genome

size. Up to 50 per cent of genes in DNA viruses are non-essential for viral replication

in permissive cells, but in non-permissive cells deletion of these immune evasion

genes results in viral clearance [189]. DNA viruses are thus able to possess a more

varied toolbox with intricate mechanisms for dealing with the Interferon Gateway

such as encoding homologues of cellular components that negatively regulate their

target such as VACV B18R, HSV 2’-5’ OA derivatives and HHV vIRF-1 and 2.

Viral Evasion and effect on lifestyle

As DNA viruses can encode more genes, this allows for a greater complexity of the

viral life style (e.g. latency) when compared to most RNA viruses that mainly cause

acute infections such as Influenza A, RABV, Ebola Virus and DFV. For example, the

HSV ICP34.5 protein is expressed early in infection before latency and integration

into the genome. Once HSV enters the lytic cycle, a different subset of genes is

expressed that includes Us11 so that the virus is still able to evade the Interferon

Gateway [190, 191]. Thus the size of DNA viral genomes allows for the temporal

regulation required for the complexity of their life cycle. This presents problems for

developing antiviral therapies against DNA viruses as they generally contain more

viral evasion proteins than RNA viruses. Adding to this difficulty is that many of the

genes of DNA viruses have not been fully characterised, which suggests that further

viral evasion proteins exist.

However, RNA viruses present an equally arduous challenge for developing antiviral

therapies. The genomes and thus the proteins of RNA viruses are constantly

mutating, due to virally encoded RNA polymerase or reverse transcriptase lacking

error correcting mechanisms [192]. DNA viral genomes do not generally undergo

such high rates of mutation as they use a cellular DNA polymerase for their

replication which has an error checking mechanism. The change in viral evasion

protein structure may also be brought about via gene segment recombination


58

between different RNA viral strains e.g. Influenza A. Additionally, selective pressure

from the antiviral therapy may promote the generation of escape mutants, and

combined with the high mutation rate of viral genomes this could lead to an alteration

in the structure of the viral evasion protein thus rendering the antiviral therapy

ineffective.

Genus and strain variation

Specific adaptations of viral evasion proteins within viral families and between

different strains can influence viral pathogenicity. Viruses within the Paramyxoviridae

family display perhaps the ultimate variation between genera, where the V, C and P

proteins encoded by the P gene are highly varied. They all share a highly conserved

C-terminus that enables them to target the STAT proteins via the W-(X)3-W-(X)9-W

Tryptophan motif or by binding to MDA5. However, different Paramyxoviruses target

different STAT proteins whilst the V proteins of the Morbillivirus genus do not [122].

The N-terminus of MeV shares only ~20% homology with other Paramyxoviruses,

and this is illustrated via a specific immune evasion mechanism of the V and C

protein forming a complex with RACK1, STAT-1 and IFNARs thereby inhibiting the

JAK kinases. Furthermore, the SeV C protein performs many of the functions of the

generic Paramyxovirus V protein. This indicates a profound evolutionary divergence

within the Paramyxoviridae family. This is not limited to Paramyxoviruses, but occurs

between different strains or subtypes of other viruses such as HPV where the nature

of the E6 protein determines the oncogenic potential of the subtype. Antiviral

strategies must therefore focus on conserved regions shared within virus families in

order to create a multi-virus vaccine or therapy.

The constant generation of novel viral strains and quasi species means that viral

evasion proteins continue to evolve to our host defences. As mentioned previously,

certain HCV strains contain the ISDR region. This is due in part to the generation of

viral quasi species in infected individuals. For example, in patients infected with the

NS5A ISDR negative HCV1b genotype, this strain evolved the ISDR region in

response to selective pressure from the Interferon Gateway [193, 194]. This is

further displayed with influenza viruses, where sequencing studies of Influenza A

NS1 have shown that its genome is inherently unstable thereby facilitating rapid

adaptation to IFN-α/β selective pressures [195]. The activity of the NS1 protein from

the 1918 pandemic strain was compared to the wild type NS1 protein in human lung

cells [196]. The pandemic strain NS1 protein was more effective than the wild type


59

NS1 protein at inhibiting the Interferon Gateway. This illustrates that future variation

in viral evasion proteins is possible and could contribute to a more deadly pathogenic

strain in future pandemics, as has happened in the past.

The findings support the “Red Queen” hypothesis where viruses and the host are

continuously developing countermeasures to gain the evolutionary upper hand [197].

The race is ongoing, where sometimes viruses develop mutations in viral proteins

that allow emerging zoonotic viruses to cross species as illustrated by recent

outbreaks of SARS-CoV, Hendra, Nipah and Ebola viruses, or the threat of

transmission of avian H5N1 influenza to humans [198]. The APOBECs and TRIM5α

ISGs restrict certain specific variants of the immunodeficiency virus to specific

species [199-203]. However, this could change if viral evasion proteins acquire the

mutations that confer the ability to evade immune restriction mechanisms. This

concept has been displayed with Feline Immunodeficiency Virus (FIV) where genetic

analysis of cheetah FIV-Ppa and leopard FIV-Aju revealed that the viruses were

closely related despite the animals evolving from different felid lineages, suggesting

recent inter-species transmission [204]. This concept could thus occur (if it has not

already) with other viruses between humans and other primates, but also with other

species such as birds, as in the case of avian influenza and SAR-CoV transmission

to humans.

The rapid rate of viral evolution compared to the vastly slower rate of human immune

systems, means that we will always face the peril of novel human pathogens

emerging from other species and the return of viruses previously successfully dealt

with by our immune systems. That is why we must create additional weapons to

enhance our armoury to counter past, present and future viral threats.

5.3. Antiviral therapies

The prospect of developing novel antivirals using viral evasion proteins as targets

has enormous potential in reducing the pathology of virus infections by aiding the

immune system to clear viral infections. In addition, antivirals could also act as a

prophylaxis to prevent further viral dissemination, which has been successfully

observed in rats with RNA interference of the RSV NSP1 gene [205]. The

conserved regions of many proteins such as the V and C proteins offer tempting

targets for drugs that would be able to inhibit a wide spectrum of Paramyxoviruses.

As mentioned previously, many RNA and DNA viral evasion proteins share the


60

ubiquitous E3 ubiquitin ligase activity. Whilst this appears to be a good target,

toxicity must be taken into account when designing antiviral therapies, to prevent

targeting of cellular pathways. This is because the viral evasion proteins may share

structural and genomic homology with cellular components.

As our molecular understanding of how viruses disrupt the Interferon Gateway has

increased, new opportunities for controlling viral infections have emerged. Thus,

attenuated virus vaccines may be developed by isolating viruses that are unable to

circumvent the IFN-α/β response. This may be achieved either by using reverse

genetics to target known genes that encode viral IFN-α/β antagonists or by selecting

mutants that are sensitive, for example Influenza A and Paramyxoviruses [206].

However, there are a number of difficulties in using IFN-α/β sensitive viruses as

vaccines. The virus may not be as attenuated as required or, alternatively, if the virus

is completely sensitive to IFN-α/β, the vaccine candidate may be over attenuated and

thus not immunogenic enough to generate an immune response and memory.

Consequently, a alternative approach may be to select for point mutations that knock

out the IFN-α/β antagonist function of the protein without affecting other functions [8].

However, single point mutations raise the possibility that such attenuated viruses

may revert to wild-type phenotypes. In addition, such IFN-α/β sensitive viruses may

be difficult to grow in culture, as most tissue-culture cells can produce and respond to

IFN-α/β following infection. Vero cells are used as they are IFN-α/β deficient, but not

all viruses can grow effectively in them [207].

The evolutionary mechanisms of DNA and RNA viruses may counter future antivirals

developed. RNA viruses have high rates of mutation leading to the generation of

many different strains, meaning that a novel strain may have an altered viral evasion

protein preventing the therapy from targeting it. HIV is notorious for its high rate of

genetic variability, forming many different quasi species in infected individuals and

successfully eluding all vaccine attempts to date [208]. Additionally susceptible

influenza viruses alter the therapeutic target by recombining with a resistant strain or

species variant, or are simply replaced by resistant strains in the population,

demonstrated by strain specific human influenza vaccines being ineffective year on

year [209]. In contrast as discussed previously, DNA viruses could acquire additional

cellular proteins that negate the effect of the antiviral. Finally, under selective

pressure RNA and DNA viruses could evolve novel strategies of evading the

Interferon Gateway and the targeted pathway, making the therapy redundant.


61

Additional Therapeutic Opportunities

Viral evasion proteins could be used as PRR agonists in order to inhibit TLR

signalling cascades that cause or contribute to acute and chronic autoimmune

diseases. In particular, TLR2 has been implicated with rheumatoid arthritis and

atherosclerosis [210], and TLR7 and 9 are thought to contribute to systemic lupus

erythematosus due to the inappropriate recognition of host nucleic acids [211]

Viral evasion proteins could also be subverted to target inflammation caused by other

factors such as injury or exposure to antigens that induce an immunopathological

response. VACV A52 derivatives are effective at reducing bacterial

lipopolysaccharide induced inflammation, liver damage and mortality in mice [212,

213]. As A52 inhibits TLR activation of NF-κB and not IRF-3 or IRF-7, use of A52-

derived peptides might preserve anti-viral immunity while inhibiting the inflammatory

response.

5.4. Conclusion

In recent years the Interferon Gateway has been uncovered as the key portal of

innate immunity. We have only just begun to understand the complex interplay

between viruses and the Interferon Gateway which could yield further drug targets as

our awareness of the arms race between viruses and host continues to grow.

However, the rapid evolution of viruses to selective pressures from the Interferon

Gateway and potential novel antiviral therapies would lead to the emergence of

resistant strains, ensuring that the arms race between humans and viruses remains

indefinite.

Word Count 13980


62

6. Acknowledgements

First and foremost I thank my supervisor Dr Andrew Macdonald for his

comprehensive support and invaluable advice. Without him this would have been

altogether a very different project! I also thank all the people from Microbial Culture

Sciences at GlaxoSmithKline during my year in industry, who gave me many

priceless tips towards writing up this project. Lastly I would also like to thank my

personal tutor Professor Keith Holland for his encouragement and perspective.


63

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8. Appendices

8.1. Abbreviations

Abbreviation Full Name

2’-5’ OAS 2'-5' Oligoadenylate Synthetase

A. a. Amino Acid

ADAR-1 Adenosine deaminase RNA 1

AIDS Acquired Immunodeficiency Syndrome

APOBEC Apolipoprotein B mRNA editing enzyme–catalytic polypeptide-like

Arg Arginine

ATP Adenosine Triphosphate

BVDV Bovine Viral Diarrhoea Virus

CARD Caspase Activation and Recruitment Domain

CBP CREB Binding Protein

CH/3 Cysteine/histidine element 3

cp Cytopathic

Cys Cysteine

DFV Dengue Fever Virus

DNA Deoxyribonucleic Acid

ds Double stranded

dsRBM dsRNA binding domain

dU Deoxyuridine

eIF2α Elongation initiation factor 2 subunit alpha

EPV Epstein-Barr Virus

FADD Fas Associated death domain

FIV Feline Immunodeficiency Virus

HAT Half a tetratricopeptide

HCV Hepatitis C Virus

Hepatitis B Virus HBV

HHV Human Herpes Virus

His Histidine

HIV Human Influenza Virus

HPV Human Papilloma Virus

HSV Herpes Simplex Virus

IBiD IRF-3 binding domain

IFNAR Type 1 Interferon Receptor


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IFN-α/β Interferon alpha/beta

IKK Inhibitor of NF-κB activator

IPS-1 IFN-β promoter stimulator 1

IRES Internal Ribosome Entry Site

IRF Interferon Regulatory Factor

ISDR Interferon Sensitivity Determining Region

ISG Interferon Stimulated Gene

ISGF3 ISG transcription factor complex IFN-stimulated gene factor 3

ISRE IFN-stimulated response element

IκB Inhibitor of NF-κB

JAK Janus Kinase

Jak1 Janus kinase 1

JEV Japanese Encephalitis Virus

JH JAK Homology domain

KIX Kinase Inducible X domain

Kα Karyopherin alpha

LGTV Langat Virus

Lys Lysine

MDA5 Melanoma differentiation associated gene 5

MeV Measles Virus

MHC Major Histocompatibility Complex

mRNA Messenger RNA

MuV Mumps Virus

MyD88 Myeloid differentiation factor 88

ncp Noncytopathic

NF-κB Nuclear Factor κB

NK Cells Natural Killer Cells

ORF Open Reading Frame

PACT PKR activating protein

PAMP Pathogen Associated Molecular Pattern

pDC Plasmoidal Dendritic Cell

PeIF2α Phosphorylated eIF2α

PHV Prospect Hill Virus

PIAS Protein inhibitor of activated STAT

PIV Parainfluenza Virus

PKR dsRNA dependent Protein Kinase R

PML Promyelocytic leukaemia


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PP1α Protein Phosphatase 1 alpha

PRMT1 Protein Arginine Methyltransferase 1

Pro Proline

PRR Pattern Recognition Receptor

PTP Protein Tyrosine Phosphatase

RABV Rabies Virus

RD Repressor Domain

RER Rough Endoplasmic Reticulum

RIG-I Retinoic acid inducible gene I

RLI RNase L Inhibitor

RNA Ribonucleic Acid

RNase L Endoribonuclease L

RSV Respiratory Syncytial Virus

SARS-CoV Severe Acute Respiratory Syndrome Coronavirus

Ser Serine

SeV Sendai Virus

SH2 Src Homology 2

SOCS Suppressors of Cytokine Signalling

ss Single stranded

STAT Signal Transducers and Activators of Transcription

TANK TRAF associated NF-κB activator

TBK TANK binding kinase

THOV Thogoto Virus

TIR Toll/Interleukin-1 Receptor

TLR Toll Like Receptors

TRAF Tumour necrosis factor receptor-associated factor

TRIF TIR domain-containing adaptor inducing IFN-β

TRIM Tripartite Motif

Tyk2 Tyrosine kinase 2

Ub Ubiquitin

VA Virus-associated RNAs

vIRF Viral IFN regulatory factor

WNV West Nile Virus

Zn Zinc

John Short Leeds Dissertation

Documents

viral disruption

viral recognition

viral replication

viral evasion proteins

viral interference of

viral interfernce of

imm short viral evasion

nature of viral inhibition