RIG-I triggers antiviral responses by recognizing viral RNA (vRNA) in the cytoplasm. However, the spatio-temporal dynamics of vRNA sensing and signal transduction remain elusive. We investigated the time course of events in cells infected with Newcastle disease virus (NDV), a non-segmented negative-strand RNA virus. RIG-I was recruited to viral replication complexes (vRC) and triggered minimal primary type I interferon (IFN) production. RIG-I subsequently localized to antiviral stress granules (avSG) induced after vRC formation. The inhibition of avSG attenuated secondary IFN production, suggesting avSG as a platform for efficient vRNA detection. avSG selectively captured positive-strand vRNA, and poly(A)+ RNA induced IFN production. Further investigations suggested that uncapped vRNA derived from read-through transcription was sensed by RIG-I in avSG. These results highlight how viral infections stimulate host stress responses, thereby selectively recruiting uncapped vRNA to avSG, in which RIG-I and other components cooperate in an efficient antiviral program.
References
[1]
Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol. 2004;5(7):730–7. doi: 10.1038/ni1087 pmid:15208624.
[2]
Baum A, Garcia-Sastre A. Induction of type I interferon by RNA viruses: cellular receptors and their substrates. Amino Acids. 2010;38(5):1283–99. Epub 2009/11/03. doi: 10.1007/s00726-009-0374-0 pmid:19882216; PubMed Central PMCID: PMCPmc2860555.
[3]
Loo Y-M, Gale M. Immune Signaling by RIG-I-like Receptors. Immunity. 2011;34(5):680–92. doi: 10.1016/j.immuni.2011.05.003 pmid:21616437.
[4]
Schlee M, Hartmann G. The chase for the RIG-I ligand—recent advances. Mol Ther. 2010;18(7):1254–62. doi: 10.1038/mt.2010.90 pmid:20461060; PubMed Central PMCID: PMCPMC2911265.
[5]
Hornung V, Ellegast J, Kim S, Brzózka K, Jung A, Kato H, et al. 5'-Triphosphate RNA is the ligand for RIG-I. Science. 2006;314(5801):994–7. doi: 10.1126/science.1132505 pmid:17038590.
[6]
Pichlmair A, Schulz O, Tan CP, N？slund TI, Liljestr？m P, Weber F, et al. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates. Science. 2006;314(5801):997–1001. doi: 10.1126/science.1132998 pmid:17038589.
[7]
Kato H, Takeuchi O, Mikamo-Satoh E, Hirai R, Kawai T, Matsushita K, et al. Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5. J Exp Med. 2008;205(7):1601–10. doi: 10.1084/jem.20080091 pmid:18591409; PubMed Central PMCID: PMCPMC2442638.
[8]
Schlee M, Roth A, Hornung V, Hagmann CA, Wimmenauer V, Barchet W, et al. Recognition of 5' triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus. Immunity. 2009;31(1):25–34. doi: 10.1016/j.immuni.2009.05.008 pmid:19576794; PubMed Central PMCID: PMCPMC2824854.
[9]
Kawai T, Takahashi K, Sato S, Coban C, Kumar H, Kato H, et al. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat Immunol. 2005;6(10):981–8. doi: 10.1038/ni1243 pmid:16127453.
[10]
Seth RB, Sun L, Ea CK, Chen ZJ. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell. 2005;122(5):669–82. doi: 10.1016/j.cell.2005.08.012 pmid:16125763.
[11]
Xu LG, Wang YY, Han KJ, Li LY, Zhai Z, Shu HB. VISA is an adapter protein required for virus-triggered IFN-beta signaling. Mol Cell. 2005;19(6):727–40. doi: 10.1016/j.molcel.2005.08.014 pmid:16153868.
[12]
Meylan E, Curran J, Hofmann K, Moradpour D, Binder M, Bartenschlager R, et al. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature. 2005;437(7062):1167–72. doi: 10.1038/nature04193 pmid:16177806.
[13]
Kedersha NL, Gupta M, Li W, Miller I, Anderson P. RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules. J Cell Biol. 1999;147(7):1431–42. pmid:10613902; PubMed Central PMCID: PMCPMC2174242. doi: 10.1083/jcb.147.7.1431
[14]
Gallouzi IE, Brennan CM, Stenberg MG, Swanson MS, Eversole A, Maizels N, et al. HuR binding to cytoplasmic mRNA is perturbed by heat shock. Proc Natl Acad Sci U S A. 2000;97(7):3073–8. pmid:10737787; PubMed Central PMCID: PMCPMC16194. doi: 10.1073/pnas.97.7.3073
[15]
Kedersha N, Chen S, Gilks N, Li W, Miller IJ, Stahl J, et al. Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules. Mol Biol Cell. 2002;13(1):195–210. doi: 10.1091/mbc.01-05-0221 pmid:11809833; PubMed Central PMCID: PMCPMC65082.
[16]
Kimball SR, Horetsky RL, Ron D, Jefferson LS, Harding HP. Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes. Am J Physiol Cell Physiol. 2003;284(2):C273–84. doi: 10.1152/ajpcell.00314.2002 pmid:12388085.
[17]
Tourrière H, Chebli K, Zekri L, Courselaud B, Blanchard JM, Bertrand E, et al. The RasGAP-associated endoribonuclease G3BP assembles stress granules. J Cell Biol. 2003;160(6):823–31. doi: 10.1083/jcb.200212128 pmid:12642610; PubMed Central PMCID: PMCPMC2173781.
[18]
Onomoto K, Yoneyama M, Fung G, Kato H, Fujita T. Antiviral innate immunity and stress granule responses. Trends Immunol. 2014;35(9):420–8. doi: 10.1016/j.it.2014.07.006 pmid:25153707.
[19]
García MA, Meurs EF, Esteban M. The dsRNA protein kinase PKR: virus and cell control. Biochimie. 2007;89(6–7):799–811. doi: 10.1016/j.biochi.2007.03.001
[20]
Onomoto K, Jogi M, Yoo JS, Narita R, Morimoto S, Takemura A, et al. Critical role of an antiviral stress granule containing RIG-I and PKR in viral detection and innate immunity. PLoS One. 2012;7(8):e43031. doi: 10.1371/journal.pone.0043031 pmid:22912779; PubMed Central PMCID: PMCPMC3418241.
[21]
Yoo JS, Takahasi K, Ng CS, Ouda R, Onomoto K, Yoneyama M, et al. DHX36 enhances RIG-I signaling by facilitating PKR-mediated antiviral stress granule formation. PLoS Pathog. 2014;10(3):e1004012. doi: 10.1371/journal.ppat.1004012 pmid:24651521; PubMed Central PMCID: PMCPMC3961341.
[22]
Lamb RA, Parks GD. Paramyxoviridae: The Viruses and Their Replication. In: Knipe DM, Howley PM, editors. Fields Virology. 1. 5 ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins; 2007. p. 1449–96.
[23]
Colonno RJ, Banerjee AK. A unique RNA species involved in initiation of vesicular stomatitis virus RNA transcription in vitro. Cell. 1976;8(2):197–204. pmid:183891. doi: 10.1016/0092-8674(76)90003-9
Loo YM, Fornek J, Crochet N, Bajwa G, Perwitasari O, Martinez-Sobrido L, et al. Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity. J Virol. 2008;82(1):335–45. doi: 10.1128/JVI.01080-07 pmid:17942531; PubMed Central PMCID: PMCPMC2224404.
[26]
Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, et al. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature. 2006;441(7089):101–5. doi: 10.1038/nature04734 pmid:16625202.
[27]
Stollar BD, Stollar V. Immunofluorescent demonstration of double-stranded RNA in the cytoplasm of Sindbis virus-infected cells. Virology. 1970;42(1):276–80. pmid:4918274. doi: 10.1016/0042-6822(70)90270-9
[28]
Gribaudo G, Lembo D, Cavallo G, Landolfo S, Lengyel P. Interferon action: binding of viral RNA to the 40-kilodalton 2'-5'-oligoadenylate synthetase in interferon-treated HeLa cells infected with encephalomyocarditis virus. J Virol. 1991;65(4):1748–57. pmid:1705989; PubMed Central PMCID: PMCPMC239981.
[29]
Lee JY, Marshall JA, Bowden DS. Characterization of rubella virus replication complexes using antibodies to double-stranded RNA. Virology. 1994;200(1):307–12. doi: 10.1006/viro.1994.1192
[30]
Westaway EG, Mackenzie JM, Kenney MT, Jones MK, Khromykh AA. Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures. J Virol. 1997;71(9):6650–61. pmid:9261387; PubMed Central PMCID: PMCPMC191943.
[31]
Zhang S, Sun Y, Chen H, Dai Y, Zhan Y, Yu S, et al. Activation of the PKR/eIF2α signaling cascade inhibits replication of Newcastle disease virus. Virol J. 2014;11:62. doi: 10.1186/1743-422X-11-62 pmid:24684861; PubMed Central PMCID: PMCPMC3994276.
[32]
Sch？nborn J, Oberstrass J, Breyel E, Tittgen J, Schumacher J, Lukacs N. Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts. Nucleic Acids Res. 1991;19(11):2993–3000. pmid:2057357; PubMed Central PMCID: PMCPMC328262. doi: 10.1093/nar/19.11.2993
[33]
Onoguchi K, Onomoto K, Takamatsu S, Jogi M, Takemura A, Morimoto S, et al. Virus-infection or 5'ppp-RNA activates antiviral signal through redistribution of IPS-1 mediated by MFN1. PLoS Pathog. 2010;6(7):e1001012. doi: 10.1371/journal.ppat.1001012 pmid:20661427; PubMed Central PMCID: PMCPMC2908619.
[34]
Matsuki H, Takahashi M, Higuchi M, Makokha GN, Oie M, Fujii M. Both G3BP1 and G3BP2 contribute to stress granule formation. Genes Cells. 2013;18(2):135–46. doi: 10.1111/gtc.12023 pmid:23279204.
[35]
Ng CS, Jogi M, Yoo JS, Onomoto K, Koike S, Iwasaki T, et al. Encephalomyocarditis virus disrupts stress granules, the critical platform for triggering antiviral innate immune responses. J Virol. 2013;87(17):9511–22. doi: 10.1128/JVI.03248-12 pmid:23785203; PubMed Central PMCID: PMCPMC3754122.
[36]
Yan Y, Samal SK. Role of intergenic sequences in newcastle disease virus RNA transcription and pathogenesis. J Virol. 2008;82(3):1323–31. doi: 10.1128/JVI.01989-07 pmid:18032502; PubMed Central PMCID: PMCPMC2224428.
[37]
Herman RC, Schubert M, Keene JD, Lazzarini RA. Polycistronic vesicular stomatitis virus RNA transcripts. Proc Natl Acad Sci U S A. 1980;77(8):4662–5. pmid:6254036; PubMed Central PMCID: PMCPMC349905. doi: 10.1073/pnas.77.8.4662
[38]
Vidal S, Kolakofsky D. Modified model for the switch from Sendai virus transcription to replication. J Virol. 1989;63(5):1951–8. pmid:2539496; PubMed Central PMCID: PMCPMC250608.
[39]
Plumet S, Duprex WP, Gerlier D. Dynamics of viral RNA synthesis during measles virus infection. J Virol. 2005;79(11):6900–8. doi: 10.1128/jvi.79.11.6900-6908.2005
[40]
Kawakami E, Watanabe T, Fujii K, Goto H, Watanabe S, Noda T, et al. Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA. J Virol Methods. 2011;173(1):1–6. doi: 10.1016/j.jviromet.2010.12.014
[41]
Plumet S, Herschke F, Bourhis JM, Valentin H, Longhi S, Gerlier D. Cytosolic 5'-triphosphate ended viral leader transcript of measles virus as activator of the RIG I-mediated interferon response. PLoS One. 2007;2(3):e279. doi: 10.1371/journal.pone.0000279
[42]
Daffis S, Szretter KJ, Schriewer J, Li J, Youn S, Errett J, et al. 2'-O methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature. 2010;468(7322):452–6. doi: 10.1038/nature09489
[43]
Schuberth-Wagner C, Ludwig J, Bruder AK, Herzner AM, Zillinger T, Goldeck M, et al. A Conserved Histidine in the RNA Sensor RIG-I Controls Immune Tolerance to N-2'O-Methylated Self RNA. Immunity. 2015. Epub 2015/07/19. doi: 10.1016/j.immuni.2015.06.015 pmid:26187414.
[44]
Runge S, Sparrer KM, L？ssig C, Hembach K, Baum A, García-Sastre A, et al. In vivo ligands of MDA5 and RIG-I in measles virus-infected cells. PLoS Pathog. 2014;10(4):e1004081. doi: 10.1371/journal.ppat.1004081 pmid:24743923; PubMed Central PMCID: PMCPMC3990713.
[45]
McAllister CS, Toth AM, Zhang P, Devaux P, Cattaneo R, Samuel CE. Mechanisms of protein kinase PKR-mediated amplification of beta interferon induction by C protein-deficient measles virus. J Virol. 2010;84(1):380–6. doi: 10.1128/JVI.02630-08 pmid:19846517; PubMed Central PMCID: PMCPMC2798421.
[46]
Li Z, Okonski KM, Samuel CE. Adenosine deaminase acting on RNA 1 (ADAR1) suppresses the induction of interferon by measles virus. J Virol. 2012;86(7):3787–94. doi: 10.1128/JVI.06307-11 pmid:22278222; PubMed Central PMCID: PMCPMC3302501.
[47]
Okonski KM, Samuel CE. Stress granule formation induced by measles virus is protein kinase PKR dependent and impaired by RNA adenosine deaminase ADAR1. J Virol. 2013;87(2):756–66. doi: 10.1128/JVI.02270-12 pmid:23115276; PubMed Central PMCID: PMCPMC3554044.
[48]
Narita R, Takahasi K, Murakami E, Hirano E, Yamamoto SP, Yoneyama M, et al. A novel function of human Pumilio proteins in cytoplasmic sensing of viral infection. PLoS Pathog. 2014;10(10):e1004417. doi: 10.1371/journal.ppat.1004417 pmid:25340845; PubMed Central PMCID: PMCPMC4207803.
[49]
Takahasi K, Yoneyama M, Nishihori T, Hirai R, Kumeta H, Narita R, et al. Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses. Mol Cell. 2008;29(4):428–40. doi: 10.1016/j.molcel.2007.11.028 pmid:18242112.
