Latest advances in innate antiviral defence.

Abstract

Recent identification of key components in the pattern recognition receptor pathway of retinoic acid-inducible gene-1-like receptors, coupled with characterisation of a new cytoplasmic DNA-sensing molecule, have led to a greater understanding of the role viral nucleic acids play in activating innate immunity. This activation of type I interferon is essential for both limiting viral infection and stimulating activation of the adaptive immune response.

Full text

Page 1

Latest advances in innate antiviral defence
Aaron Irving and Bryan RG Williams*
Address: Monash Institute of Medical Research, Monash University, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia
*Corresponding author: Bryan RG Williams (bryan.williams@med.monash.edu.au)
F1000 Biology Reports 2009, 1:22 (doi: 10.3410/B1-22)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial License
(http://creativecommons.org/licenses/by-nc/3.0/legalcode), which permits unrestricted use, distribution, and reproduction in any medium,
The electronic version of this article is the complete one and can be found at: http://F1000.com/Reports/Biology/content/1/22
Abstract
Recent identification of key components in the pattern recognition receptor pathway of retinoic acid-
inducible gene-1-like receptors, coupled with the characterisation of a new cytoplasmic DNA-sensing
molecule, has led to a greater understanding of the role that viral nucleic acids play in activating innate
immunity. This activation of type-I interferon is essential for both limiting viral infection and
stimulating activation of the adaptive immune response.
Introduction and context
Upon gaining access to a cell, a virus is recognised by the
innate immune response of the host, predominantly
through the presence of foreign nucleic acids binding to
pattern recognition receptors (PRRs). Key effectors of this
response are Toll-like receptors (TLRs) and retinoic acid-
inducible gene-1 (RIG-I)-like receptors (RLRs), which act
by triggering signalling cascades, ultimately resulting in
the production of type-I interferons (IFNs), the corre-
sponding interferon-stimulated genes (ISGs), and activa-
tion of other key regulators of innate immunity, such as
nuclear factor-kappa-B (NF-kB). This initial response is
essential to limit viral infection and activate natural killer
cells and dendritic cells, setting in train the adaptive
immune response. While much has been learned about
TLR signalling pathways, the mechanism by which RLRs
lead to IFN activation has yet to be fully elucidated. The
recent independent discovery by different groups of a key
regulator named MPYS, stimulator of interferon genes
(STING), or MITA has now identified a critical compo-
nent of the pathway linking RLRs to type-I IFN
production [1,2]. Moreover, other studies have revealed
a key component in the mechanism of DNA-dependent
activation of the IFN regulatory factors (IRFs) [3].
RIG-I, melanoma differentiation-associated gene 5
(MDA5), and laboratory of genetics and physiology 2
(LGP2) are a small family of RNA helicases residing in
the cytoplasm that contain RNA-binding domains
capable of recognising foreign viral transcripts, such as
those from negative-strand RNA viruses [4]. Whereas all
three helicases function similarly, they differ in size-
specific and sequence motif-specific recognition of viral
transcripts [5,6]. RIG-I also contains a self-regulatory
domain that recognises 50-triphosphorylated single-
stranded or double-stranded RNA to allow activation
[7]. This affects the ability of each helicase to activate the
innate immune system in response to different patho-
gens. The activation occurs through the tandem CARD
(caspase recruitment domain) in RIG-I and MDA5,
which binds to the mitochondrial antiviral signalling
protein (MAVS/IPS-1/CARDIF/VISA) [8–11]. Once acti-
vated, MAVS triggers activation of two protein complexes
[TANK-binding kinase 1 (TBK1)–IkB kinase epsilon
(IKKe)–IKKg–TANK (TRAF family member-associated
NF-kB activator) and IKKa–IKKb–IKKg] involved in
activation of the IRFs and the NF-kB transcription factor,
respectively (Figure 1).
Major recent advances
Two research groups have independently identified a
transmembrane protein, residing in both the endoplas-
mic reticulum and mitochondrial membrane, which is
necessary for RIG-I-mediated IFN activation [1,2]. While
differences in localisation were reported in the two
papers, it was proposed that STING interacted with a
RIG-I-SSR2/TRAPb translocon complex, facilitating a link
between viral RNA transcripts and IFN activation. Zhong
Page 1 of 4
(page number not for citation purposes)
Published: 17 March 2009
© 2009 Biology Reports Ltd
for non-commercial purposes provided the original work is properly cited. You may not use this work for commercial purposes.

Page 2

et al. [2] also showed a link for STING in RIG-I signalling,
through MAVS, and its interaction with TBK1/IRF3.
STING is encoded by the identical gene previously
named MPYS, a plasma membrane tetraspanner
implicated in mitochondrial and surface membrane
presentation of antigens through an interaction with
major histocompatibility complex type II [12]. However,
an unequivocal role for this molecule has been shown in
pathogen-associated molecular pattern recognition
through the use of knockout mice. In mice deficient in
STING or in cells derived from these mice, IRF3
activation via TBK1 is compromised and an increased
susceptibility to viral infections is evident. However,
there also appears to be a cell-type dependency of STING
for production of a complete IFN response to viral RNA.
Despite differences in the predicted mechanism of
action, one via MAVS and the other via the translocon
complex, both groups also showed an additional role for
STING in non-CpG DNA-mediated induction of type-I
IFNs.
In addition to activating an RNA-sensing mechanism,
virus DNA can induce a protective innate immune
response through other cellular sensors. This recognition
was believed to be through the action of TLR9 recognis-
ing CpG-rich DNA [13,14]. However, there is also
evidence indicating a TLR9-independent activator of
ISGs [15]. Recent work has revealed DNA-dependent
activator of interferon regulatory factors DAI/DLM-1/
ZBP1 as a key component in the recognition of DNA
through its three DNA-binding domains. DNA binding
by DAI activates dimer formation enabling interaction
with TBK1 and IRF3, enhancing activation of IRF3 and
possibly IRF7 in response to foreign cytoplasmic DNA.
This interaction is dependent upon DNA to maintain the
interaction of DAI with TBK1 [16].
Interestingly, the ability of DAI to induce IRF activation
in response to pathogenic or host DNA residing in the
cytoplasm is cell type-dependent [16]. In mouse
embryonic fibroblasts with depleted DAI, there was
only a minimal reduction to non-CpG (B form)-
mediated DNA induction of DNA. This, along with
previous evidence, suggests that other DNA-sensing
molecules required for recognition of DNA present in
the cytoplasm remain to be identified. Nonetheless,
reduction of DAI in the macrophage cell lines showed a
decrease in IFN induction upon viral infection.
The characterisation of further components in the RLR
signalling pathway has identified an important link
between cytoplasmic RNA sensors and the TBK1/IRF
protein complex for activation of IFN. The DNA
cytoplasmic sensor, DAI, also interacts with TBK1/IRF.
This major component of IFN induction plays a role in
both RNA and DNA cytoplasmic sensing and emphasises
a convergence of two different PRR pathways.
Figure 1. Cytoplasmic recognition of viral nucleic acids
Stimulation of DAI and possibly other DNA sensors by viral dsDNA (or
B-form DNA) activates the TANK-NAP1-SINTBAD-IKKg-IKKe-TBK1
kinase complex and stimulates phosphorylation of IRF3 and probably IRF7.
This process could explain the function of STING/MITA/MPYS in DNA
sensing. Viral ss/dsRNA recognition through RIG-I-MAVS interaction
requiresSTINGandtheTRAP-Sec61-Exocystcomplextostimulatethesame
kinase complex as DNA sensing and also activates the IKKg-IKKa-IKKb
complex to trigger NF-kB activation. DAI, DNA-dependent activator of
interferonregulatoryfactor; ds,double-stranded;ER,endoplasmicreticulum;
IFN-b, interferon (type I)-beta; IKK, IkB kinase; IRF, interferon regulatory
factor; MAVS, mitochondrial antiviral signalling protein; NAP1, nuclear
factor-kappa-B-activating kinase-associated protein 1; NF-kB, nuclear factor-
kappa-B; P, phosphate; RIG-I, retinoic acid-inducible gene-1; SINTBAD,
similar to nuclear factor-kappa-B-activating kinase-associated protein 1
TANK (TRAF family member-associated nuclear factor-kappa-B activator)-
binding kinase 1 adaptor; ss, single-stranded; STING, stimulator of interferon
genes; TANK, TRAF family member-associated nuclear factor-kappa-B
activator; TBK1, TANK (TRAF family member-associated nuclear factor-
kappa-B activator)-binding kinase 1; TRAP, translocon-associated protein.
Page 2 of 4
(page number not for citation purposes)
F1000 Biology Reports 2009, 1:22http://F1000.com/Reports/Biology/content/1/22

Page 3

Important to note is the abundance of viral proteins
aimed at inhibiting either RLR sensing (for example,
influenza virus NS1 [17,18] and human metapneumo-
virus G protein [19]) or DNA sensing, inhibited by
Vaccinia virus E3L [20] and porcine circovirus type 2
[21]. Different viral proteins have also evolved to target
the convergence of the two pathways and inhibit TBK1
and IRF3 phosphorylation. These include Ebola virus
VP35 [22], Herpes simplex virus 1 g34.5 [23], rabies
virus P protein [24], and hantavirus G1 [25], among
many others (reviewed in [26]).
Future directions
These recent findings provide new insights into the
signalling pathways triggered by viral nucleic acids but
still leave many unanswered questions. The exact
mechanism of action for STING/MITA remains to be
determined, along with its localisation and role in either
MAVS activation or the translocon complex. The latter
raises questions about the way viral nucleic acids are
processed and presented to activate the innate immune
response of the cell.
Further investigation should lead to the identification of
other cytoplasmic DNA sensors and elucidate which
sensors are required in each cellular compartment or cell
type. This will provide a better understanding of host
response to different pathogens and of the particular cell
type engaged by the host to recognise the infection.
Careful characterisation of all components required for
recognising cellular pathogens will lead to a greater
understanding of the innate immune response and
ideally provide new therapeutic targets to help treat
infections.
Abbreviations
DAI, DNA-dependent activator of interferon regulatory
factor; IFN, interferon (type I); IKK, IkB kinase; IRF,
interferon regulatory factor; ISG, interferon-stimulated
gene; MAVS, mitochondrial antiviral signalling protein;
MDA5, melanoma differentiation-associated gene 5; NF-
kB, nuclear factor-kappa-B; PRR, pattern recognition
receptor; RIG-I, retinoic acid-inducible gene-1; RLR,
retinoic acid-inducible gene-1-like receptor; STING,
stimulator of interferon genes; TANK, TRAF family
member-associated nuclear factor-kappa-B activator;
TBK1, TANK (TRAF family member-associated nuclear
factor-kappa-B activator)-binding kinase 1; TLR, Toll-like
receptor.
Competing interests
The authors declare that they have no competing
interests.
Acknowledgements
Work in the authors’ laboratory is supported by grants
from the US National Institutes of Health (P01
CA062220 and R01 AI034039) and the Australian
National Health and Medical Research Council
(436814).
References
1. Ishikawa H, Barber GN: STING is an endoplasmic reticulum
adaptor that facilitates innate immune signalling. Nature 2008,
455:674-8.
F1000 Factor 9.9 Exceptional
Evaluated by Bryan Williams 8 Sep 2008, Michael Gale 9 Sep 2008,
Grant McFadden 10 Oct 2008, Adolfo Garcia-Sastre 13 Oct 2008
2. Zhong B, Yang Y, Li S, Wang YY, Li Y, Diao F, Lei C, He X, Zhang L,
Tien P, Shu HB: The adaptor protein MITA links virus-sensing
receptors to IRF3 transcription factor activation. Immunity
2008, 29:538-50.
F1000 Factor 6.0 Must Read
Evaluated by Grant McFadden 24 Oct 2008
3. Takaoka A, Wang Z, Choi MK, Yanai H, Negishi H, Ban T, Lu Y,
Miyagishi M, Kodama T, Honda K, Ohba Y, Taniguchi T: DAI (DLM-
1/ZBP1) is a cytosolic DNA sensor and an activator of innate
immune response. Nature 2007, 448:501-5.
F1000 Factor 6.5 Must Read
Evaluated by Alan Houghton 2 Aug 2007, Grant McFadden 3 Aug
2007, David Underhill 7 Aug 2007
4.Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T,
Miyagishi M, Taira K, Akira S, Fujita T: The RNA helicase RIG-I has
an essential function in double-stranded RNA-induced innate
antiviral responses. Nat Immunol 2004, 5:730-7.
F1000 Factor 8.0 Exceptional
Evaluated by Grant McFadden 2 Jul 2004, Caetano Reis e Sousa 14
Jul 2004
5. Kato H, Takeuchi O, Mikamo-Satoh E, Hirai R, Kawai T, Matsushita K,
Hiiragi A, Dermody TS, Fujita T, Akira S: Length-dependent
recognition of double-stranded ribonucleic acids by retinoic
acid-inducible gene-I and melanoma differentiation-asso-
ciated gene 5. J Exp Med 2008, 205:1601-10.
F1000 Factor 3.0 Recommended
Evaluated by Caetano Reis e Sousa
6.Saito T, Owen DM, Jiang F, Marcotrigiano J, Gale M Jr.: Innate
immunity induced by composition-dependent RIG-I recogni-
tion of hepatitis C virus RNA. Nature 2008, 454:523-7.
F1000 Factor 4.8 Must Read
Evaluated by Bryan Williams 17 Jun 2008, Grant McFadden 30 Jul
2008
7.Takahasi K, Yoneyama M, Nishihori T, Hirai R, Kumeta H, Narita R,
Gale M Jr., Inagaki F, Fujita T: Nonself RNA-sensing mechanism
of RIG-I helicase and activation of antiviral immune
responses. Mol Cell 2008, 29:428-40.
Kawai T, Takahashi K, Sato S, Coban C, Kumar H, Kato H, Ishii KJ,
Takeuchi O, Akira S: IPS-1, an adaptor triggering RIG-I- and
Mda5-mediated type I interferon induction. Nat Immunol 2005,
6:981-8.
Meylan E, Curran J, Hofmann K, Moradpour D, Binder M,
Bartenschlager R, Tschopp J: Cardif is an adaptor protein in the
RIG-I antiviral pathway and is targeted by hepatitis C virus.
Nature 2005, 437:1167-72.
8.
9.
F1000 Factor 8.0 Exceptional
Evaluated by Astar Winoto 29 Sep 2005, Eckhard Podack 2 Dec
2005
Page 3 of 4
(page number not for citation purposes)
F1000 Biology Reports 2009, 1:22 http://F1000.com/Reports/Biology/content/1/22

Page 4

10. Seth RB, Sun L, Ea CK, Chen ZJ: Identification and characteriza-
tion of MAVS, a mitochondrial antiviral signaling protein that
activates NF-kappaB and IRF 3. Cell 2005, 122:669-82.
F1000 Factor 9.8 Exceptional
Evaluated by David Wallach 6 Sep 2005, John Kyriakis 28 Sep 2005,
Jenny Ting 11 Nov 2005
11. Xu LG, Wang YY, Han KJ, Li LY, Zhai Z, Shu HB: VISA is an
adapter protein required for virus-triggered IFN-beta signal-
ing. Mol Cell 2005, 19:727-40.
Jin L, Waterman PM, Jonscher KR, Short CM, Reisdorph NA, Cambier
JC: MPYS, a novel membrane tetraspanner, is associated with
major histocompatibility complex class II and mediates
transduction of apoptotic signals. Mol Cell Biol 2008, 28:5014-26.
Ishii Y, Yamaguchi K; Meropenem Surveillance Group: Evaluation of
the susceptibility trends to meropenem in a nationwide
collection of clinical isolates in Japan: a longitudinal analysis
from 2002 to 2006. Diagn Microbiol Infect Dis 2008, 61:346-50.
Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R,
Koretzky GA, Klinman DM: CpG motifs in bacterial DNA trigger
direct B-cell activation. Nature 1995, 374:546-9.
Ishii KJ, Coban C, Kato H, Takahashi K, Torii Y, Takeshita F, Ludwig H,
SutterG,SuzukiK,HemmiH,SatoS,YamamotoM,UematsuS,KawaiT,
Takeuchi O, Akira S: A Toll-like receptor-independent antiviral
responseinduced by double-strandedB-form DNA. Nat Immunol
2006, 7:40-8.
12.
13.
14.
15.
F1000 Factor Exceptional
Evaluated by Caetano Reis e Sousa 23 Nov 2005, Grant McFadden
24 Jan 2006
16.Wang Z, Choi MK, Ban T, Yanai H, Negishi H, Lu Y, Tamura T,
Takaoka A, Nishikura K, Taniguchi T: Regulation of innate
immune responses by DAI (DLM-1/ZBP1) and other DNA-
sensing molecules. Proc Natl Acad Sci U S A 2008, 105:5477-82.
F1000 Factor 6.0 Must Read
Evaluated by Grant McFadden 9 Apr 2008
17.Min JY, Krug RM: The primary function of RNA binding by the
influenza A virus NS1 protein in infected cells: inhibiting the
20-50oligo (A) synthetase/RNase L pathway. Proc Natl Acad Sci U
S A 2006, 103:7100-5.
Pichlmair A, Schulz O, Tan CP, Naslund TI, Liljestrom P, Weber F,
Reis e Sousa C: RIG-I-mediated antiviral responses to single-
18.
stranded
314:997-1001.
RNA bearing50-phosphates.
Science2006,
F1000 Factor 8.3 Exceptional
Evalauted by Jurg Tschopp 27 Oct 2006, Patrick S. Moore 21 Nov
2006, Clifford Harding 24 Nov 2006, Rino Rappuoli 28 Nov 2006
19.Biacchesi S, Skiadopoulos MH, Yang L, Lamirande EW, Tran KC,
Murphy BR, Collins PL, Buchholz UJ: Recombinant human
Metapneumovirus lacking the small hydrophobic SH and/or
attachment G glycoprotein: deletion of G yields a promising
vaccine candidate. J Virol 2004, 78:12877-87.
Smith EJ, Marie I, Prakash A, Garcia-Sastre A, Levy DE: IRF3 and
IRF7 phosphorylation in virus-infected cells does not require
double-stranded RNA-dependent protein kinase R or Ikappa
B kinase but is blocked by Vaccinia virus E3L protein. J Biol
Chem 2001, 276:8951-7.
Wikstrom FH, Meehan BM, Berg M, Timmusk S, Elving J, Fuxler L,
Magnusson M, Allan GM, McNeilly F, Fossum C: Structure-
dependent modulation of alpha interferon production by
porcine circovirus 2 oligodeoxyribonucleotide and CpG
DNAs in porcine peripheral blood mononuclear cells. J Virol
2007, 81:4919-27.
Cardenas WB, Loo YM, Gale M Jr., Hartman AL, Kimberlin CR,
Martinez-Sobrido L, Saphire EO, Basler CF: Ebola virus VP35
protein binds double-stranded RNA and inhibits alpha/beta
interferon production induced by RIG-I signaling. J Virol 2006,
80:5168-78.
Verpooten D, Ma Y, Hou S, Yan Z, He B: Control of TANK-
binding kinase 1 mediated signaling by the gamma134.5
protein of herpes simplex virus 1. J Biol Chem 2009,
284:1097-105.
Brzozka K, Finke S, Conzelmann KK: Identification of the rabies
virus alpha/beta interferon antagonist: phosphoprotein P
interferes with phosphorylation of interferon regulatory
factor 3. J Virol 2005, 79:7673-81.
Alff PJ, Gavrilovskaya IN, Gorbunova E, Endriss K, Chong Y,
Geimonen E, Sen N, Reich NC, Mackow ER: The pathogenic
NY-1 hantavirus G1 cytoplasmic tail inhibits RIG-I- and TBK-
1-directed interferon responses. J Virol 2006, 80:9676-86.
Bowie AG, Unterholzner L: Viral evasion and subversion of
pattern-recognition receptor signalling. Nat Rev Immunol 2008,
8:911-22.
20.
21.
22.
23.
24.
25.
26.
Page 4 of 4
(page number not for citation purposes)
F1000 Biology Reports 2009, 1:22 http://F1000.com/Reports/Biology/content/1/22