Structural and biochemical studies of RIG-I antiviral signaling

Abstract

Retinoic acid-inducible gene I (RIG-I) is an important pattern recognition receptor that detects viral RNA and triggers the production of type-I interferons through the downstream adaptor MAVS (also called IPS-1, CARDIF, or VISA). A series of structural studies have elaborated some of the mechanisms of dsRNA recognition and activation of RIG-I. Recent studies have proposed that K63-linked ubiquitination of, or unanchored K63-linked polyubiquitin binding to RIG-I positively regulates MAVS-mediated antiviral signaling. Conversely phosphorylation of RIG-I appears to play an inhibitory role in controlling RIG-I antiviral signal transduction. Here we performed a combined structural and biochemical study to further define the regulatory features of RIG-I signaling. ATP and dsRNA binding triggered dimerization of RIG-I with conformational rearrangements of the tandem CARD domains. Full length RIG-I appeared to form a complex with dsRNA in a 2:2 molar ratio. Compared with the previously reported crystal structures of RIG-I in inactive state, our electron microscopic structure of full length RIG-I in complex with blunt-ended dsRNA, for the first time, revealed an exposed active conformation of the CARD domains. Moreover, we found that purified recombinant RIG-I proteins could bind to the CARD domain of MAVS independently of dsRNA, while S8E and T170E phosphorylation-mimicking mutants of RIG-I were defective in binding E3 ligase TRIM25, unanchored K63-linked polyubiquitin, and MAVS regardless of dsRNA. These findings suggested that phosphorylation of RIG inhibited downstream signaling by impairing RIG-I binding with polyubiquitin and its interaction with MAVS.

Keywords

RIG-I / MAVS / antiviral signaling / polyubiquitin / phosphorylation

References

[1] Arimoto, K., Takahashi, H., Hishiki, T., Konishi, H., Fujita, T., and Shimotohno, K. (2007). Negative regulation of the RIG-I signaling by the ubiquitin ligase RNF125. Proc Natl Acad Sci U S A 104, 7500-7505 .10.1073/pnas.0611551104
[2] Chacon, P., and Wriggers, W. (2002). Multi-resolution contour-based fitting of macromolecular structures. J Mol Biol 317, 375-384 .10.1006/jmbi.2002.5438
[3] Cong, Y., Baker, M.L., Jakana, J., Woolford, D., Miller, E.J., Reissmann, S., Kumar, R.N., Redding-Johanson, A.M., Batth, T.S., and Mukhopadhyay, A. (2010). 4.0-? resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement. Proc Natl Acad Sci U S A 107, 4967-4972 .10.1073/pnas.0913774107
[4] Cong, Y., and Ludtke, S.J. (2010). Single particle analysis at high resolution. Methods Enzymol 482, 211-235 .10.1016/S0076-6879(10)82009-9
[5] Gack, M.U., Kirchhofer, A., Shin, Y.C., Inn, K.S., Liang, C., Cui, S., Myong, S., Ha, T., Hopfner, K.P., and Jung, J.U. (2008). Roles of RIG-I N-terminal tandem CARD and splice variant in TRIM25-mediated antiviral signal transduction. Proc Natl Acad Sci U S A 105, 16743-16748 .10.1073/pnas.0804947105
[6] Gack, M.U., Nistal-Villan, E., Inn, K.S., Garcia-Sastre, A., and Jung, J.U. (2010). Phosphorylation-mediated negative regulation of RIG-I antiviral activity. J Virol 84, 3220-3229 .10.1128/JVI.02241-09
[7] Gack, M.U., Shin, Y.C., Joo, C.H., Urano, T., Liang, C., Sun, L., Takeuchi, O., Akira, S., Chen, Z., and Inoue, S. (2007). TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446, 916-920 .10.1038/nature05732
[8] Hornung, V., Ellegast, J., Kim, S., Brzozka, K., Jung, A., Kato, H., Poeck, H., Akira, S., Conzelmann, K.K., Schlee, M., . (2006). 5′-Triphosphate RNA is the ligand for RIG-I. Science 314, 994-997 .10.1126/science.1132505
[9] Hou, F., Sun, L., Zheng, H., Skaug, B., Jiang, Q.X., and Chen, Z.J. (2011). MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response. Cell 146, 448-461 .10.1016/j.cell.2011.06.041
[10] Janeway, C.A., Jr., and Medzhitov, R. (2002). Innate immune recognition. Annu Rev Immunol 20, 197-216 .10.1146/annurev.immunol.20.083001.084359
[11] Jiang, F., Ramanathan, A., Miller, M.T., Tang, G.Q., Gale, M., Jr., Patel, S.S., and M Ramanathan arcotrigiano, J. (2011). Structural basis of RNA recognition and activation by innate immune receptor RIG-I. Nature 479, 423-427 .10.1038/nature10537
[12] Jiang, X., Kinch, L.N., Brautigam, C.A., Chen, X., Du, F., Grishin, N.V., and Chen, Z.J. (2012). Ubiquitin-Induced Oligomerization of the RNA Sensors RIG-I and MDA5 Activates Antiviral Innate Immune Response. Immunity 36, 959-973 .10.1016/j.immuni.2012.03.022
[13] Kato, H., Takeuchi, O., Mikamo-Satoh, E., Hirai, R., Kawai, T., Matsushita, K., Hiiragi, A., Dermody, T.S., Fujita, T., and Akira, S. (2008). Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5. J Exp Med 205, 1601-1610 .10.1084/jem.20080091
[14] Kawai, T., Takahashi, K., Sato, S., Coban, C., Kumar, H., Kato, H., Ishii, K.J., Takeuchi, O., and Akira, S. (2005). IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat Immunol 6, 981-988 .10.1038/ni1243
[15] Kowalinski, E., Lunardi, T., McCarthy, A.A., Louber, J., Brunel, J., Grigorov, B., Gerlier, D., and Cusack, S. (2011). Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA. Cell 147, 423-435 .10.1016/j.cell.2011.09.039
[16] Kumar, H., Kawai, T., and Akira, S. (2009). Pathogen recognition in the innate immune response. Biochem J 420, 1-16 .10.1042/BJ20090272
[17] Leung, D.W., and Amarasinghe, G.K. (2012). Structural insights into RNA recognition and activation of RIG-I-like receptors. Curr Opin Struct Biol 22, 297-303 10.1016/j.sbi.2012.03.011
[18] Liu, H.M., Loo, Y.M., Horner, S.M., Zornetzer, G.A., Katze, M.G., and Gale, M., Jr. (2012). The mitochondrial targeting chaperone 14-3-3epsilon regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity. Cell Host Microbe 11, 528-537 .10.1016/j.chom.2012.04.006
[19] Lu, C., Ranjith-Kumar, C.T., Hao, L., Kao, C.C., and Li, P. (2011). Crystal structure of RIG-I C-terminal domain bound to blunt-ended double-strand RNA without 5′ triphosphate. Nucleic Acids Res 39, 1565-1575 .10.1093/nar/gkq974
[20] Lu, C., Xu, H., Ranjith-Kumar, C.T., Brooks, M.T., Hou, T.Y., Hu, F., Herr, A.B., Strong, R.K., Kao, C.C., and Li, P. (2010). The structural basis of 5′ triphosphate double-stranded RNA recognition by RIG-I C-terminal domain. Structure 18, 1032-1043 .10.1016/j.str.2010.05.007
[21] Ludtke, S.J., Baldwin, P.R., and Chiu, W. (1999). EMAN: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128, 82-97 .10.1006/jsbi.1999.4174
[22] Ludtke, S.J., Jakana, J., Song, J.L., Chuang, D.T., and Chiu, W. (2001). A 11.5 A single particle reconstruction of GroEL using EMAN. J Mol Biol 314, 253-262 .10.1006/jmbi.2001.5133
[23] Luo, D., Kohlway, A., Vela, A., and Pyle, A.M. (2012). Visualizing the Determinants of Viral RNA Recognition by Innate Immune Sensor RIG-I. Structure 20, 1983-1988 .10.1016/j.str.2012.08.029
[24] Maharaj, N.P., Wies, E., Stoll, A., and Gack, M.U. (2012). Conventional protein kinase C-alpha (PKC-alpha) and PKC-beta negatively regulate RIG-I antiviral signal transduction. J Virol 86, 1358-1371 .10.1128/JVI.06543-11
[25] Meylan, E., Curran, J., Hofmann, K., Moradpour, D., Binder, M., Bartenschlager, R., and Tschopp, J. (2005). Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, 1167-1172 .10.1038/nature04193
[26] Nistal-Villan, E., Gack, M.U., Martinez-Delgado, G., Maharaj, N.P., Inn, K.S., Yang, H., Wang, R., Aggarwal, A.K., Jung, J.U., and Garcia-Sastre, A. (2010). Negative role of RIG-I serine 8 phosphorylation in the regulation of interferon-beta production. J Biol Chem 285, 20252-20261 .10.1074/jbc.M109.089912
[27] Oh, S.D., Lao, J.P., Hwang, P.Y., Taylor, A.F., Smith, G.R., and Hunter, N. (2007). BLM ortholog, Sgs1, prevents aberrant crossing-over by suppressing formation of multichromatid joint molecules. Cell 130, 259-272 .10.1016/j.cell.2007.05.035
[28] Oshiumi, H., Matsumoto, M., Hatakeyama, S., and Seya, T. (2009). Riplet/RNF135, a RING finger protein, ubiquitinates RIG-I to promote interferon-beta induction during the early phase of viral infection. J Biol Chem 284, 807-817 .10.1074/jbc.M804259200
[29] Oshiumi, H., Miyashita, M., Inoue, N., Okabe, M., Matsumoto, M., and Seya, T. (2010). The ubiquitin ligase Riplet is essential for RIG-I-dependent innate immune responses to RNA virus infection. Cell Host Microbe 8, 496-509 .10.1016/j.chom.2010.11.008
[30] Pichlmair, A., Schulz, O., Tan, C.P., Naslund, T.I., Liljestrom, P., Weber, F., and Reis e Sousa, C. (2006). RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates. Science 314, 997-1001 .10.1126/science.1132998
[31] Saito, T., Hirai, R., Loo, Y.M., Owen, D., Johnson, C.L., Sinha, S.C., Akira, S., Fujita, T., and Gale Jr, M. (2007). Regulation of innate antiviral defenses through a shared repressor domain in RIG-I and LGP2. Science′s STKE 104, 582.
[32] Schlee, M., Roth, A., Hornung, V., Hagmann, C.A., Wimmenauer, V., Barchet, W., Coch, C., Janke, M., Mihailovic, A., Wardle, G., . (2009). Recognition of 5′ triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus. Immunity 31, 25-34 .10.1016/j.immuni.2009.05.008
[33] Takahasi, K., Yoneyama, M., Nishihori, T., Hirai, R., Kumeta, H., Narita, R., Gale, M., Jr., Inagaki, F., and Fujita, T. (2008). Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses. Mol Cell 29, 428-440 .10.1016/j.molcel.2007.11.028
[34] Takeuchi, O., and Akira, S. (2010). Pattern recognition receptors and inflammation. Cell 140, 805-820 .10.1016/j.cell.2010.01.022
[35] Tang, G., Peng, L., Baldwin, P.R., Mann, D.S., Jiang, W., Rees, I., and Ludtke, S.J. (2007). EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol 157, 38-46 .10.1016/j.jsb.2006.05.009
[36] Wriggers, W. (2012). Conventions and workflows for using Situs. Acta Crystallogr D Biol Crystallogr 68, 344-351 .10.1107/S0907444911049791
[37] Wriggers, W., Milligan, R.A., and McCammon, J.A. (1999). Situs: A package for docking crystal structures into low-resolution maps from electron microscopy. J Struct Biol 125, 185-195 .10.1006/jsbi.1998.4080
[38] Xu, L.G., Wang, Y.Y., Han, K.J., Li, L.Y., Zhai, Z., and Shu, H.B. (2005). VISA is an adapter protein required for virus-triggered IFN-beta signaling. Mol Cell 19, 727-740 .10.1016/j.molcel.2005.08.014
[39] Yoneyama, M., Kikuchi, M., Matsumoto, K., Imaizumi, T., Miyagishi, M., Taira, K., Foy, E., Loo, Y.M., Gale, M.,Jr., Akira, S., . (2005). Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity. J Immunol 175, 2851-2858 .
[40] Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., Taira, K., Akira, S., and Fujita, T. (2004). The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol 5, 730-737 .10.1038/ni1087
[41] Zeng, W., Sun, L., Jiang, X., Chen, X., Hou, F., Adhikari, A., Xu, M., and Chen, Z.J. (2010). Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity. Cell 141, 315-330 .10.1016/j.cell.2010.03.029