Function of RIG-I-like Receptors in Antiviral Innate Immunity

Various cells in the body are capable of sensing infectious viruses and initiating reactions collectively known as antiviral innate responses. These responses include the production of antiviral cytokines such as type I interferon (IFN) 2The abbreviations used are: IFN, interferon; RIG-I, retinoic acid-inducible gene I; RLR, RIG-I-like receptor(s); dsRNA, double-stranded RNA; ssRNA, single-stranded RNA; CARD, caspase recruitment domain; IPS-1, interferon promoter stimulator-1; TRAF3, TNF receptor-associating factor 3; IKK-i, IκB kinase-i; TBK-1, TANK-binding kinase-1; cDC, conventional dendritic cell; NDV, Newcastle disease virus; pDC, plasmacytoid dendritic cell; IL, interleukin; TLR, Toll-like receptor; MyD88, myeloid differentiation factor 88; IRAK1, interleukin-1 receptor-associated kinase 1; IRF, interferon regulatory factor; VSV, vesicular stomatitis virus; JEV, Japanese encephalitis virus; EMCV, encephalomyocarditis virus; RNAi, RNA interference. and subsequent synthesis of antiviral enzymes, which are responsible for the impairment of viral replication and promoting adaptive immune responses (1Samuel C.E. Clin. Microbiol. Rev. 2001; 14: 778-809Crossref PubMed Scopus (2168) Google Scholar). In this minireview, we focus on a subset of molecules known as RIG-I-like receptors, which sense viral RNA molecules that trigger a danger signal. Three genes encode RIG-I-like receptors (RLR) in human and mouse genomes (2Yoneyama M. Kikuchi M. Matsumoto K. Imaizumi T. Miyagishi M. Taira K. Foy E. Loo Y.M. Gale Jr., M. Akira S. Yonehara S. Kato A. Fujita T. J. Immunol. 2005; 175: 2851-2858Crossref PubMed Scopus (1302) Google Scholar). Three DExD/H box helicases, termed retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated antigen 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), exhibit marked primary structure conservation in their helicase domain (Fig. 1). As the analysis of RIG-I precedes the other two, the biochemical characteristics of RIG-I will be described in this section. As shown in Fig. 1, RIG-I contains two repeats of the caspase recruitment domain (CARD)-like motif at its N terminus. The cDNA clone was initially obtained by functional screening based on reporter gene activation, essentially consisting of tandem CARD (3Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3136) Google Scholar). Although less efficient than the signal by fulllength RIG-I activated by viral infection, overexpression of the tandem CARD alone is sufficient to generate signaling and subsequent type I IFN production (2Yoneyama M. Kikuchi M. Matsumoto K. Imaizumi T. Miyagishi M. Taira K. Foy E. Loo Y.M. Gale Jr., M. Akira S. Yonehara S. Kato A. Fujita T. J. Immunol. 2005; 175: 2851-2858Crossref PubMed Scopus (1302) Google Scholar). CARD acts as a signaling domain, which interacts with a downstream molecule (IPS-1, see below) to relay the signal. Single amino acid substitution within the first CARD (T55I) is sufficient to inactivate CARD function, and tandem CARD is necessary for its function (4Sumpter Jr., R. Loo Y.M. Foy E. Li K. Yoneyama M. Fujita T. Lemon S.M. Gale Jr., M. J. Virol. 2005; 79: 2689-2699Crossref PubMed Scopus (734) Google Scholar). So far there is no report showing the signal-dependent proteolytic release of CARD from full-length RIG-I, suggesting that processing is unlikely in the mechanism of RIG-I activation. Fulllength RIG-I exhibits undetectable or very low constitutive activity in the cell transfection assay, suggesting that the C-terminal region contains a domain for autorepression. Indeed, functional analysis revealed that the C-terminal domain (Fig. 1, Repression Domain) is responsible for autorepression by interacting with both CARD and helicase domains (5Saito T. Hirai R. Loo Y.M. Owen D. Johnson C.L. Sinha S.C. Akira S. Fujita T. Gale Jr., M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 582-587Crossref PubMed Scopus (586) Google Scholar). Interestingly, RIG-I with loss of function of CARD, either by deletion (RIG-IC) or point mutation (T55I), is incapable of transmitting a signal upon viral infection and dominantly inhibits virus-induced signaling (3Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3136) Google Scholar, 4Sumpter Jr., R. Loo Y.M. Foy E. Li K. Yoneyama M. Fujita T. Lemon S.M. Gale Jr., M. J. Virol. 2005; 79: 2689-2699Crossref PubMed Scopus (734) Google Scholar). This is because of the lack of a signaling domain and the presence of a repression domain as well as RNA binding activity. RIG-I exhibits strong double-stranded RNA (dsRNA) binding activity in vitro. RIG-I selectively binds with poly(rI:rC), poly(rA:rU), and 5′- and 3′-untranslated regions of hepatitis C virus genomic RNA (which are predicted to form a secondary structure) but not with dsDNA, poly(rA), or yeast tRNA (3Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3136) Google Scholar, 4Sumpter Jr., R. Loo Y.M. Foy E. Li K. Yoneyama M. Fujita T. Lemon S.M. Gale Jr., M. J. Virol. 2005; 79: 2689-2699Crossref PubMed Scopus (734) Google Scholar). RNA binding requires intact helicase and C-terminal autorepression domains (5Saito T. Hirai R. Loo Y.M. Owen D. Johnson C.L. Sinha S.C. Akira S. Fujita T. Gale Jr., M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 582-587Crossref PubMed Scopus (586) Google Scholar) (Fig. 1). The above results suggest that RIG-I is a specific sensor for dsRNA, which is absent in uninfected cells but known to be accumulated in virus-infected cells; however, influenza A virus infection results in IFN gene activation without detectable dsRNA accumulation (6Pichlmair A. Schulz O. Tan C.P. Naslund T.I. Liljestrom P. Weber F. Reis e Sousa C. Science. 2006; 314: 997-1001Crossref PubMed Scopus (1779) Google Scholar). In these cells, it is proposed that single-stranded RNA (ssRNA) with 5′-triphosphate functions as a ligand for RIG-I. Actually RIG-I specifically binds with RNA containing 5′-triphosphate but not with RNA containing 5′-di- or 5′-monophosphate (7Hornung V. Ellegast J. Kim S. Brzozka K. Jung A. Kato H. Poeck H. Akira S. Conzelmann K.K. Schlee M. Endres S. Hartmann G. Science. 2006; 314: 994-997Crossref PubMed Scopus (1909) Google Scholar). These observations led to an interesting hypothesis of how self and non-self RNA species are discriminated. As shown in Fig. 2, host RNA synthesis takes place in the nucleus. Like the viral transcript, cellular primary transcripts contain 5′-triphosphate; however, these RNAs undergo various processes; mRNA acquires a 7-methylguanosine CAP structure at its 5′-end; tRNA undergoes 5′-cleavage and a series of nucleotide base modifications; the primary transcript of ribosomal RNA readily complexes with ribosomal proteins to form ribosomal ribonucleoprotein and undergoes maturation processes, which therefore are masked from detection. Indeed, artificial capping and base modification of 5′-triphosphate ssRNA abrogated detection by RIG-I (7Hornung V. Ellegast J. Kim S. Brzozka K. Jung A. Kato H. Poeck H. Akira S. Conzelmann K.K. Schlee M. Endres S. Hartmann G. Science. 2006; 314: 994-997Crossref PubMed Scopus (1909) Google Scholar), whereas viral RNA, either freshly introduced by infection or produced by viral replication, contains a non-self marker, 5′-triphosphate. In this regard, 5′-triphosphate RNA generated by DNA virus may well be detected by RIG-I. It is worth noting that a single amino acid substitution K270A renders RIG-I into a dominant inhibitor (3Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3136) Google Scholar). Lys-270 is supposed to be a critical motif for ATP binding within the helicase domain, and in the case of other DExH/D helicases, this motif is crucial for its helicase (unwinding dsRNA) activity. As proteolysis is an unlikely mechanism (above) to reverse autorepression, the current de-repression model for RIG-I is illustrated in Fig. 3. RIG-I exists as a “closed” structure in uninfected cells and therefore CARD is masked. The virus-specific RNA species, dsRNA or 5′-triphosphate ssRNA, specifically binds to RIG-I through its RNA binding domain. This association and ATP binding to the helicase domain change RIG-I conformation to release CARD for relaying signaling to the downstream molecule (another CARD-containing molecule, IPS-1 (alternatively termed MAVS, VISA, and Cardif)) (8Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. Ishii K.J. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 981-988Crossref PubMed Scopus (2032) Google Scholar, 9Seth R.B. Sun L. Ea C.K. Chen Z.J. Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2518) Google Scholar, 10Xu L.G. Wang Y.Y. Han K.J. Li L.Y. Zhai Z. Shu H.B. Mol. Cell. 2005; 19: 727-740Abstract Full Text Full Text PDF PubMed Scopus (1518) Google Scholar, 11Meylan E. Curran J. Hofmann K. Moradpour D. Binder M. Bartenschlager R. Tschopp J. Nature. 2005; 437: 1167-1172Crossref PubMed Scopus (1973) Google Scholar). IPS-1 is localized on the outer membrane of mitochondria, and this localization is crucial for its function (9Seth R.B. Sun L. Ea C.K. Chen Z.J. Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2518) Google Scholar, 12Loo Y.M. Owen D.M. Li K. Erickson A.K. Johnson C.L. Fish P.M. Carney D.S. Wang T. Ishida H. Yoneyama M. Fujita T. Saito T. Lee W.M. Hagedorn C.H. Lau D.T. Weinman S.A. Lemon S.M. Gale Jr., M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6001-6006Crossref PubMed Scopus (351) Google Scholar, 13Lin R. Lacoste J. Nakhaei P. Sun Q. Yang L. Paz S. Wilkinson P. Julkunen I. Vitour D. Meurs E. Hiscott J. J. Virol. 2006; 80: 6072-6083Crossref PubMed Scopus (193) Google Scholar, 14Li X.D. Sun L. Seth R.B. Pineda G. Chen Z.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17717-17722Crossref PubMed Scopus (662) Google Scholar) although its precise mechanism is not known. MDA5 and RIG-I, which sense a distinct set of viruses (below), commonly transmit signals to IPS-1; thus IPS-1–/– fibroblasts are unresponsive to either set of viruses (15Kumar H. Kawai T. Kato H. Sato S. Takahashi K. Coban C. Yamamoto M. 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Med. 2004; 199: 1641-1650Crossref PubMed Scopus (465) Google Scholar), which is responsible for the activation of IRF-3 and -7. LGP2 lacks CARD, suggesting that this helicase is incapable of transmitting a positive signal. Overexpression of LGP2 in cell culture results in the dominant inhibition of virus-induced activation of IFN genes, suggesting its role as a negative regulator; however, its role in vivo is not established (2Yoneyama M. Kikuchi M. Matsumoto K. Imaizumi T. Miyagishi M. Taira K. Foy E. Loo Y.M. Gale Jr., M. Akira S. Yonehara S. Kato A. Fujita T. J. Immunol. 2005; 175: 2851-2858Crossref PubMed Scopus (1302) Google Scholar, 20Rothenfusser S. Goutagny N. DiPerna G. Gong M. Monks B.G. Schoenemeyer A. Yamamoto M. Akira S. Fitzgerald K.A. J. Immunol. 2005; 175: 5260-5268Crossref PubMed Scopus (490) Google Scholar, 21Komuro A. Horvath C.M. J. Virol. 2006; 80: 12332-12342Crossref PubMed Scopus (245) Google Scholar) (Table 1).TABLE 1Differential functions of RIG-I family helicasesRIG-IPositive regulatorNDV, Sendai virus, influenza virus, VSV, JEV, in vitro transcribed dsRNAMDA5Positive regulatorPicornavirus, poly (I):poly (C)LGP2Negative regulator?? Open table in a new tab Analyses of RIG-I–/– fibroblasts and conventional dendritic cells (cDCs) showed that RIG-I is essential in Newcastle disease virus (NDV)-induced IFN production; however, RIG-I is dispensable for virus-induced IFN production by plasmacytoid dendritic cells (pDCs) (22Kato H. Sato S. Yoneyama M. Yamamoto M. Uematsu S. Matsui K. Tsujimura T. Takeda K. Fujita T. Takeuchi O. Akira S. Immunity. 2005; 23: 19-28Abstract Full Text Full Text PDF PubMed Scopus (1118) Google Scholar). pDCs adopt a distinct signaling cascade to produce high levels of IFN-α and sense viral infection by TLR7/8 and TLR9, activating signaling cascades MyD88, IRAK1/4, TRAF3/6, IKK-α, and IRF-7 (23Takeda K. Akira S. Int. Immunol. 2005; 17: 1-14Crossref PubMed Scopus (2731) Google Scholar, 24Honda K. Taniguchi T. Nat. Rev. Immunol. 2006; 6: 644-658Crossref PubMed Scopus (1280) Google Scholar). It has been known that dsRNA activates TLR3 in endosome and signals through TIR domain-containing adaptor inducing IFN-β (TRIF)/TIR-containing adaptor molecule-1 (TICAM1) resulting in the activation of kinases (TBK-1 or IKK-i) and transcription factors (IRF-3 and -7 and NF-κB) (23Takeda K. Akira S. Int. Immunol. 2005; 17: 1-14Crossref PubMed Scopus (2731) Google Scholar). However, the mice defective in the TLR3-TRIF pathway exhibit normal IFN response upon viral infections (25Kato H. Takeuchi O. Sato S. Yoneyama M. Yamamoto M. Matsui K. Uematsu S. Jung A. Kawai T. Ishii K.J. Yamaguchi O. Otsu K. Tsujimura T. Koh C.S. Reis e Sousa C. Matsuura Y. Fujita T. Akira S. Nature. 2006; 441: 101-105Crossref PubMed Scopus (2933) Google Scholar). When poly(rI:rC) is injected into mice intravenously, IFN-β is strictly produced in a MDA5-dependent manner (below), but the TLR-TRIF pathway is dispensable. However, production of IL-8 and IL-12 p40 requires TRIF in addition to MDA5; IL-12 p40 is particularly largely TRIF-dependent (25Kato H. Takeuchi O. Sato S. Yoneyama M. Yamamoto M. Matsui K. Uematsu S. Jung A. Kawai T. Ishii K.J. Yamaguchi O. Otsu K. Tsujimura T. Koh C.S. Reis e Sousa C. Matsuura Y. Fujita T. Akira S. Nature. 2006; 441: 101-105Crossref PubMed Scopus (2933) Google Scholar). These observations demonstrate that although MDA5 and TLR3 signal a common pathway, different spectrums of cytokine genes are activated. The overall structural similarity between RIG-I and MDA5 suggests the functional similarity of these proteins. Gene disruption studies revealed that these helicases sense distinct viral species (25Kato H. Takeuchi O. Sato S. Yoneyama M. Yamamoto M. Matsui K. Uematsu S. Jung A. Kawai T. Ishii K.J. Yamaguchi O. Otsu K. Tsujimura T. Koh C.S. Reis e Sousa C. Matsuura Y. Fujita T. Akira S. Nature. 2006; 441: 101-105Crossref PubMed Scopus (2933) Google Scholar). Cytokine production induced by the infection of Sendai virus, NDV, vesicular stomatitis virus (VSV), influenza A virus, and Japanese encephalitis virus (JEV) is markedly impaired in RIG-I–/– cells (Table 1). In contrast, cytokine production by encephalomyocarditis virus (EMCV), Thyler's virus and Mengo virus, all Picornaviruses (genus cardiovirus), is virtually absent in MDA5–/– cells (Table 1). In agreement with these observations, virus challenge experiments using knockout mice revealed that RIG-I–/– and MDA5–/– mice are selectively vulnerable to JEV and EMCV, respectively. It is remarkable that RIG-I/MDA5 deficiency exhibits a severe impact on viral infection in vivo, suggesting the critical function of innate immune responses in promoting adaptive immunity and virus eradication. Interestingly, genomic RNA of VSV and poly(rI:rC) selectively activates RIG-I and MDA5, respectively, suggesting that the distinct responses of RIG-I and MDA5 to different viruses are because of the distinct recognition of viral RNA by these sensors. Viruses evolve to avoid host immune surveillance by producing inhibitors of the IFN system (Table 2). Generally, viral replication takes place in a restricted compartment where the viral genome is protected from detection by host sensors. Mouse hepatitis virus takes this strategy to avoid innate immune responses (26Zhou H. Perlman S. J. Virol. 2007; 81: 568-574Crossref PubMed Scopus (98) Google Scholar). Viral proteins evolve to counteract RLR functions. NS3/4A of hepatitis C virus inactivates IPS-1 by its protease activity (9Seth R.B. Sun L. Ea C.K. Chen Z.J. Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2518) Google Scholar, 12Loo Y.M. Owen D.M. Li K. Erickson A.K. Johnson C.L. Fish P.M. Carney D.S. Wang T. Ishida H. Yoneyama M. Fujita T. Saito T. Lee W.M. Hagedorn C.H. Lau D.T. Weinman S.A. Lemon S.M. Gale Jr., M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6001-6006Crossref PubMed Scopus (351) Google Scholar, 13Lin R. Lacoste J. Nakhaei P. Sun Q. Yang L. Paz S. Wilkinson P. Julkunen I. Vitour D. Meurs E. Hiscott J. J. Virol. 2006; 80: 6072-6083Crossref PubMed Scopus (193) Google Scholar, 14Li X.D. Sun L. Seth R.B. Pineda G. Chen Z.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17717-17722Crossref PubMed Scopus (662) Google Scholar). Other viral proteins inhibit RLR signaling at various steps for their survival. It is noteworthy that many proteins encoded by DNA viruses also target RLR signaling.TABLE 2Viral inhibitors of RLR signalingVirusInhibitorMode of actionRefs.Hepatitis C virusNS3/4ACleavage of IPS-19Seth R.B. Sun L. Ea C.K. Chen Z.J. Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2518) Google Scholar, 12Loo Y.M. Owen D.M. Li K. Erickson A.K. Johnson C.L. Fish P.M. Carney D.S. Wang T. Ishida H. Yoneyama M. Fujita T. Saito T. Lee W.M. Hagedorn C.H. Lau D.T. Weinman S.A. Lemon S.M. Gale Jr., M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6001-6006Crossref PubMed Scopus (351) Google Scholar, 13Lin R. Lacoste J. Nakhaei P. Sun Q. Yang L. Paz S. Wilkinson P. Julkunen I. Vitour D. Meurs E. Hiscott J. J. Virol. 2006; 80: 6072-6083Crossref PubMed Scopus (193) Google Scholar, 14Li X.D. Sun L. Seth R.B. Pineda G. Chen Z.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17717-17722Crossref PubMed Scopus (662) Google ScholarInfluenza A VirusNS1dsRNA binding, binding with RIG-I and IPS-16Pichlmair A. Schulz O. Tan C.P. Naslund T.I. Liljestrom P. Weber F. Reis e Sousa C. Science. 2006; 314: 997-1001Crossref PubMed Scopus (1779) Google Scholar, 31Mibayashi M. Martinez-Sobrido L. Loo Y.M. Cardenas W.B. Gale Jr., M. Garcia-Sastre A. J. Virol. 2007; 81: 514-524Crossref PubMed Scopus (499) Google Scholar, 32Opitz B. Rejaibi A. Dauber B. Eckhard J. Vinzing M. Schmeck B. Hippenstiel S. Suttorp N. Wolff T. Cell Microbiol. 2007; 9: 930-938Crossref PubMed Scopus (231) Google Scholar, 33Guo Z. Chen L.M. Zeng H. Gomez J.A. Plowden J. Fujita T. Katz S. J. Cell Mol. 2007; PubMed Scopus Google binding, inhibition of TBK-1, W.B. Loo Y.M. Gale Jr., M. Martinez-Sobrido L. J. Virol. 2006; 80: PubMed Scopus Google to M. Kikuchi M. Matsumoto K. Imaizumi T. Miyagishi M. Taira K. Foy E. Loo Y.M. Gale Jr., M. Akira S. Yonehara S. Kato A. Fujita T. J. 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Full Text Full Text PDF PubMed Scopus Google however, no has been on the role of RIG-I or MDA5 in RNAi, suggesting that structural similarity may be a of is of the IFN system in cells M. Nat. Cell 5: PubMed Scopus Google Scholar). It has been that gene either by transfection of dsRNA or of RNA has the to IFN gene and downstream M. Nat. Cell 5: PubMed Scopus Google Scholar, S. A. R. Nat. PubMed Scopus Google Scholar). In this regard, the of structure of RNA is T. Wang D. M. P. Hartmann R. Fujita T. Nat. 2006; 24: PubMed Scopus Google however, for the activation of RLR pathway and to be