1. ABSTRACT 1
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.
Viral Evasion of the Interferon Gateway
John A. L. Short and Dr. Andrew Macdonald
Faculty of Biological Sciences, University of Leeds, LS2 9JT
P
STAT-2
P
STAT-1
Cell membrane
P
STAT-1
P
STAT-2
IFNAR2
STAT-1
IFNAR1
Tyk2 Jak1
Nipah V Protein
Hendra V Protein
Rabies Virus P Protein
STAT-1
P
Measles Virus P protein
Sendai Virus C protein
Hepatitis C Virus NS5A protein
Sendai Virus C protein
Hepatitis C Virus Core protein
Tyk2 Jak1
STAT-2
STAT-1
P STAT-2
PSTAT-1
Nucleus
ISGs
IRF-9
ISRE
IRF-9
ISGF3
STAT-2
PSTAT-1
P
BA
DISCUSSION
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.
Clearly, viruses that had not evolved IFN-α/β evasion strategies would now be extinct.
Consequently both RNA and DNA viruses have developed an impressive array of mechanisms to
surmount all levels of the Interferon Gateway.
INTRODUCTION
Viruses and their hosts have a dynamic relationship, constantly evolving strategies to outwit
the other in a battle for survival.
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. Its role is to either clear the infection or hold it at bay
until an adaptive response is mounted. This is mediated in part by Interferons (IFN) (Fig.1).
Fig. 1. The Innate Immune System Matrix.
Green dashed arrows indicate the target of cytokines
produced. Pink dashed arrows show the target
of IFN-α/β produced.
Viral Nucleic Acids
TLR Pathway RIG-I/MDA5 Pathways
JAK/STAT Pathway
Interferon
Stimulated
Genes
RNA Viruses
Bovine Viral Diarrhoea Virus
Ebola Virus
Influenza A
Rotavirus
DNA Viruses
Epstein Barr
Herpes Simplex
Vaccinia
Transcription Factor
Activation Pathway
RNA Viruses
Hepatitis C Virus
DNA Viruses
Vaccina
RNA Viruses
Hepatitis C Virus
Influenza A
Measles Virus
Mumps Virus
Parainfluenza
Respiratory
Syncytial Virus
Sedai Virus
RNA Viruses
Borna Disease Virus
Bovine Viral Diarrhoea Virus
Classical Swine Fever Virus
Ebola Virus
Influenza A
NY-1V
Rabies Virus
Respiratory Syncytial Virus
Rotavirus
SARS-CoV
Thogoto Virus
DNA Viruses
Adenovirus
Human Herpes Virus 6
Human Herpes Virus 8
Herpes Simplex
RNA Viruses
Hepatitis C Virus
Hendra Virus
Japanese Encephalitis Virus
Langat virus
Measles Virus
Mumps Virus
Nipah Virus
Parainfluenza
RABV
SARS-CoV
Sendai Virus
West Nile Virus
DNA Viruses
Hepatitis B Virus
Human Cytomegalovirus
Human Papilloma Virus
Herpes Simplex
Vaccinia
RNA Viruses
Hepatitis C Virus
HIV
Rabies Virus
DNA Viruses
Adenovirus
Epstein Barr
Human Herpes Virus 8
Human Papilloma Virus
Herpes Simplex
Vaccinia
Viral Inhibition of the JAK/STAT Pathway 5
Virus
Virus Infected Cell
TLRs RIG-I MDA5
Interferon Stimulated Genes
JAK/STAT Pathway
Blood/ Tissues
Inflammation
Macrophages pDCComplement
Adaptive Immune System
NK Cells
Virus Infected Cell Apoptosis
PKR 2'-5' OAS MxADAR-1
TRIMs,
APOBECs
PML
Extracellular Immune Responses Intracellular Immune Responses
RNase L
• What are Interferons?
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.
• What is the Interferon Gateway?
The gateway is the generation of the cellular
antiviral state mediated by the expression
and subsequent action of Type I alpha and
beta interferons (IFN-α/β).
IFN-α/β acts in an auto-, para- and endocrine
manner, subsequently inducing the
expression of Interferon Stimulated Genes
(ISGs) in infected and neighbouring
uninfected cells.
ISG products inhibit viral replication by
inducing the degradation of viral RNA or can
induce apoptosis by the shutdown of
translation if the cell is overwhelmed,
mediated by RNase L & Protein Kinase R
(PKR) respectively.
• Why is the Interferon Gateway important?
The Interferon Gateway is the lynchpin of the
innate host defence against virus infection.
Without it, viruses would completely
overwhelm the host before the adaptive
immune system had a chance to respond.
AIMS 3
• Viruses have evolved strategies to actively evade and subvert the Interferon Gateway at all
stages.
• Many viruses have adapted by expressing viral proteins that act as “keys”, modulating the
Interferon Gateway by “locking” or inhibiting multiple levels to enable continued 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 to develop novel antivirals.
Conclusion 8
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.
Acknowledgements
I thank Dr. Andrew Macdonald for his comprehensive guidance and support. I also thank Professor
Keith Holland for his encouragement and perspective.
Key References
Meylan, E., J. Tschopp, and M. Karin, Intracellular pattern recognition receptors in the host response. Nature, 2006. 442(7098): p. 39-44.
Pichlmair, A. and C. Reis e Sousa, Innate recognition of viruses. Immunity, 2007. 27(3): p. 370-83.
Randall, R.E. and S. Goodbourn, Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures. J Gen
Virol, 2008. 89(Pt 1): p. 1-47.
Unterholzner, L. and A.G. Bowie, The interplay between viruses and innate immune signaling: Recent insights and therapeutic opportunities. Biochem
Pharmacol, 2008. 75(3): p. 589-602.
Viral Inhibition of Interferon Stimulated Genes: PKR 6
• PKR is a seronine threonine kinase
that phosphorylates Elongation initiation
factor 2 subunit alpha (eIF2α).
• eIF2α recruits tMET to ribosomes to
form a translation pre-initiation complex.
• Phosphorylated eIF2α irreversibly
binds to the nucleotide exchange factor
eIF2B, “freezing” eIF2α in the complex
preventing it from initiating future
translational events.
• Translation of viral and cellular
proteins is blocked.
• Viruses have adapted by evolving a
variety of mechanisms to inhibit PKR at
all stages in the pathway.
PKR
PKR
PKR
dsRNA
P
P
eIF2
P
eIF2
Inactive for
Initiation of Translation
Active for
Initiation of Translation
Active
Kinase
Inactive
Kinase
Viral dsRNA homologues
Adenovirus E1A
Epstein Barr EB1
Hepatitis C Virus Internal
Ribosome Entry Site
Mediators of eIF2
Dephosphorylation
Human Papilloma Virus E6
Herpes Simplex ICP34.5
PKR Binding Proteins
Hepatitis C Virus E2
Hepatitis C Virus NS5A
HIV Tat
Herpes Simplex Us11
Influenza A NS1
Vaccinia E3L
Vaccinia K3L
dsRNA binding Proteins
Ebola Virus VP35
Epstein Barr EB2
Herpes Simplex Us11
Influenza A NS1
Rotavirus Sigma3
Vaccinia E3L
• IFN-α/β binds to the type I IFN receptors (IFNAR)
which then form a heterodimer.
• IFNARs are associated with Tyk2 and Jak1
kinases that phosphorylate the STAT proteins.
• Phosphorylated STAT proteins form a
heterodimer that translocates to the nucleus,
forming the IFN-stimulated gene factor 3
complex (ISGF3) that induces expression of the
ISGs.
• Viral evasion proteins can act as STAT protein
binding platforms, sequestering:
1. inactive STAT proteins from phosphorylation by
the JAK kinases.
2. active STAT heterodimers from translocating to
the nucleus in a degradation independent
manner , thus preventing ISG expression.
Fig. 3. Viral STAT Protein Sequestration
Strategies. A) Viral inhibition of STAT
heterodimer formation. B) The cellular
normal pathway.
Fig. 4. Viral PKR
Inhibition Strategies.
A) RIG-I/ MDA5 Pathway
• Many viruses utilise the cytosol, either for
genome replication or intracellular transport.
• Unsurprisingly, the host has evolved cytosolic
detectors of viral nucleic acids, that initiate a
signal transduction pathway culminating in the
transcription of IFN-α/β.
• Paramyxoviruses encode V and C proteins that
bind to the helicase domains of MDA5 and RIG-I
respectively, sequestering them from viral
nucleic acids.
• Influenza A NS1 protein binds to the CARD
domains of RIG-I and IPS-1 forming an inactive
complex.
• Hepatitis C Virus NS3/4A is a serine threonine
kinase that cleaves IPS-1 from mitochondria,
redistributing it to the cytosol where it is inactive.
B) TLR3/4 Pathway
• Many viruses exploit the endocytic system
during their life-cycle for initial infection of the
cell and also for egress of virions containing
newly replicated genomes
• To prevent virus subversion the host has evolved a
class of sentinel TLRs that reside in the endocytic
system.
• Vaccinia Virus encodes the A46 and A52 viral
proteins that bind to the TIR domains,
preventing TLR signalling components from
interacting with each other.
• Hepatitis C Virus NS3/4A cleaves the TBK-1
binding domain from TRIF, preventing the
TBK-1 recruitment.
Viral Inhibition of Interferon-α/β Expression 4
Fig. 2. Viral IFN-α/β Expression Inhibition Strategies.
A) Activated Melanoma differentiation associated gene 5 (MDA5) and Retinoic acid
inducible gene I (MDA5/RIG-I) initiate signal transduction via adaptor protein
domains called Caspase Activation and Recruitment Domain (CARDs) that interact
with homologous CARDs found on downstream signalling components
B) Activated Toll Like Receptors (TLRs) 3/4 initiate signal transduction via adaptor
protein domains called Toll/Interleukin-1 Receptor (TIR) that are analogous to the
CARD domains.
Endosome
TLR3
TLR4
MyD88
TRIF
= TIR Domain
Cell Membrane
TRAF6
TRAF6 Complex
IRF-3
P
c-Jun
P P
ATF-2
P
NS3/4A
TRAM MAL
A52
A46
Key
RIG-I
TRAF6/FADD Complex
Helicase Domain Helicase Domain
MDA5
Mitochondrion
IPS-1
V C
NS1
NS3/4A
= CARDDomain
P
IRF-7
P
TBK-1 Complex
Cytosol
P = Phosphate
Nucleus
IRF-7
P
IRF-7
P
c-Jun
P P
ATF-2 IRF-3
P
IRF-3
P
RIG-I/MDA5 Pathway TLR3/4 Pathway
Viral dsRNA
Viral
dsRNA
Viral envelope Glycoproteins
Viral ssRNA
= Interferon Transcription
Factor
= Viral Evasion Protein
= Phosphorylation
A B
2
7
Fig. 5. 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.
• 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 expression, signalling
and ISG effecter pathways by converging on key
signalling mediators e.g. IPS-1.
• RNA viruses, despite having smaller genomes
than DNA viruses, are equally capable of
inhibiting the Interferon Gateway. Both DNA and
RNA viral evasion proteins use conserved
functions to target many levels of the gateway.
This is due to an equilibrium of two contrasting
selective pressures:
1. Constant downward pressure on genome size.
2. Selective pressures applied by Interferon
Gateway to adapt viral proteins for evasion.
• Specific adaptations of viral evasion proteins
within viral families and strains can influence
pathogenicity.
• The constant generation of novel viral strains
and quasi species means that viral evasion
proteins continue to evolve to our host defences.
• The findings support the “Red Queen”
hypothesis where viruses and the host are
continuously developing countermeasures to
gain the evolutionary upper hand.
• 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.
• The development of novel antivirals is
essential to enhance our armoury to counter
past, present and future viral threats.
Therapeutic Opportunities
• Novel antivirals using viral evasion proteins
as targets has enormous potential in reducing
the pathology of virus infections
• Antivirals could act as a prophylaxis to
prevent further viral dissemination.
• Viral evasion proteins could be subverted for
reducing the immunopathology caused by
autoimmune diseases or other factors e.g.
tissue injury, Toxic Shock Syndrome.
Influenza A
Taken from: www.3DScience.com
IFN-
eIF2
IKK Complex
IB NF-B
NF-BIB
IKK Complex
NF-B
IFN-
IFN-
IFN-Promoter
IFN- Promoter
IFN-/IFN-/
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