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Transcript of 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
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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].
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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).
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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).
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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).
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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.
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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].
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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.
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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].
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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.
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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,
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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.
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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
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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].
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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
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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.
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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].
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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).
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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
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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).
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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.
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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
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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
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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
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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.
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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
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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).
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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
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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
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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.
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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
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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
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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
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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
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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].
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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
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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.
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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).
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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.
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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.
14th April 2008 Viral Evasion of the Interferon Gateway John A L Short
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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
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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.
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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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Abbreviation Full Name
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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Abbreviation Full Name
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