Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지
DOI QR코드

DOI QR Code

Hepatitis C Virus Nonstructural Protein 5A Interacts with Immunomodulatory Kinase IKKε to Negatively Regulate Innate Antiviral Immunity

  • Kang, Sang-Min (Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University) ;
  • Park, Ji-Young (Division of Chronic Viral Disease, Center for Emerging Virus Research, National Institute of Infectious Diseases, National Institute of Health, Korea Disease Control and Prevention Agency) ;
  • Han, Hee-Jeong (Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University) ;
  • Song, Byeong-Min (Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University) ;
  • Tark, Dongseob (Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University) ;
  • Choi, Byeong-Sun (Division of Chronic Viral Disease, Center for Emerging Virus Research, National Institute of Infectious Diseases, National Institute of Health, Korea Disease Control and Prevention Agency) ;
  • Hwang, Soon B. (Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University)
  • Received : 2022.02.02
  • Accepted : 2022.05.23
  • Published : 2022.10.31
Download PDF
⟨ Previous Next ⟩

Abstract

Hepatitis C virus (HCV) infection can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV employs diverse strategies to evade host antiviral innate immune responses to mediate a persistent infection. In the present study, we show that nonstructural protein 5A (NS5A) interacts with an NF-κB inhibitor immunomodulatory kinase, IKKε, and subsequently downregulates beta interferon (IFN-β) promoter activity. We further demonstrate that NS5A inhibits DDX3-mediated IKKε and interferon regulatory factor 3 (IRF3) phosphorylation. We also note that hyperphosphorylation of NS5A mediates protein interplay between NS5A and IKKε, thereby contributing to NS5A mediated modulation of IFN-β signaling. Lastly, NS5A inhibits IKKε-dependent p65 phosphorylation and NF-κB activation. Based on these findings, we propose NS5A as a novel regulator of IFN signaling events, specifically by inhibiting IKKε downstream signaling cascades through its interaction with IKKε. Taken together, these data suggest an additional mechanistic means by which HCV modulates host antiviral innate immune responses to promote persistent viral infection.

Keywords

Acknowledgement

This work was supported by grant from the Korea National Institute of Health (2014-NG51001 and 2016-NG51005 for S.M.K.). This work was also supported by the NRF grant funded by the Korea government (MSIT) (2021R1A2C2003275 for S.B.H.).

References

  1. Adli, M. and Baldwin, A.S. (2006). IKK-i/IKKepsilon controls constitutive, cancer cell-associated NF-kappaB activity via regulation of Ser-536 p65/ RelA phosphorylation. J. Biol. Chem. 281, 26976-26984. https://doi.org/10.1074/jbc.M603133200
  2. Appel, N., Pietschmann, T., and Bartenschlager, R. (2005). Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain. J. Virol. 79, 3187-3194. https://doi.org/10.1128/JVI.79.5.3187-3194.2005
  3. Appel, N., Zayas, M., Miller, S., Krijnse-Locker, J., Schaller, T., Friebe, P., Kallis, S., Engel, U., and Bartenschlager, R. (2008). Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLoS Pathog. 4, e1000035. https://doi.org/10.1371/journal.ppat.1000035
  4. Asabe, S.I., Tanji, Y., Satoh, S., Kaneko, T., Kimura, K., and Shimotohno, K. (1997). The N-terminal region of hepatitis C virus-encoded NS5A is important for NS4A-dependent phosphorylation. J. Virol. 71, 790-796. https://doi.org/10.1128/jvi.71.1.790-796.1997
  5. Balka, K.R., Louis, C., Saunders, T.L., Smith, A.M., Calleja, D.J., D'Silva, D.B., Moghaddas, F., Tailler, M., Lawlor, K.E., Zhan, Y., et al. (2020). TBK1 and IKKε act redundantly to mediate STING-induced NF-κB responses in myeloid cells. Cell Rep. 31, 107492. https://doi.org/10.1016/j.celrep.2020.03.056
  6. Brownell, J., Bruckner, J., Wagoner, J., Thomas, E., Loo, Y.M., Gale, M., Jr., Liang, T.J., and Polyak, S.J. (2014). Direct, interferon-independent activation of the CXCL10 promoter by NF-kappaB and interferon regulatory factor 3 during hepatitis C virus infection. J. Virol. 88, 1582-1590. https://doi.org/10.1128/JVI.02007-13
  7. Choi, J.W., Kim, J.W., Nguyen, L.P., Nguyen, H.C., Park, E.M., Choi, D.H., Han, K.M., Kang, S.M., Tark, D., Lim, Y.S., et al. (2020). Nonstructural NS5A protein regulates LIM and SH3 domain protein 1 to promote hepatitis C virus propagation. Mol. Cells 43, 469-478.
  8. Choi, S.H. and Hwang, S.B. (2006). Modulation of TGF-beta signal transduction pathway by hepatitis C virus nonstructural 5A protein. J. Biol. Chem. 281, 7468-7478. https://doi.org/10.1074/jbc.M512438200
  9. Ferreon, J.C., Ferreon, A.C., Li, K., and Lemon, S.M. (2005). Molecular determinants of TRIF proteolysis mediated by the hepatitis C virus NS3/4A protease. J. Biol. Chem. 280, 20483-20492. https://doi.org/10.1074/jbc.M500422200
  10. Fitzgerald, K.A., McWhirter, S.M., Faia, K.L., Rowe, D.C., Latz, E., Golenbock, D.T., Coyle, A.J., Liao, S.M., and Maniatis, T. (2003). IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4, 491-496.
  11. Gale, M., Jr., Blakely, C.M., Kwieciszewski, B., Tan, S.L., Dossett, M., Tang, N.M., Korth, M.J., Polyak, S.J., Gretch, D.R., and Katze, M.G. (1998). Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation. Mol. Cell. Biol. 18, 5208-5218. https://doi.org/10.1128/MCB.18.9.5208
  12. Gale, M.J., Jr., Korth, M.J., Tang, N.M., Tan, S.L., Hopkins, D.A., Dever, T.E., Polyak, S.J., Gretch, D.R., and Katze, M.G. (1997). Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 230, 217-227. https://doi.org/10.1006/viro.1997.8493
  13. Gu, L., Fullam, A., Brennan, R., and Schroder, M. (2013). Human DEAD box helicase 3 couples IkappaB kinase epsilon to interferon regulatory factor 3 activation. Mol. Cell. Biol. 33, 2004-2015. https://doi.org/10.1128/MCB.01603-12
  14. Hoofnagle, J.H. (2002). Course and outcome of hepatitis C. Hepatology 36(5 Suppl 1), S21-S29.
  15. Indukuri, H., Castro, S.M., Liao, S.M., Feeney, L.A., Dorsch, M., Coyle, A.J., Garofalo, R.P., Brasier, A.R., and Casola, A. (2006). Ikkepsilon regulates viral-induced interferon regulatory factor-3 activation via a redox-sensitive pathway. Virology 353, 155-165. https://doi.org/10.1016/j.virol.2006.05.022
  16. Jensen, S. and Thomsen, A.R. (2012). Sensing of RNA viruses: a review of innate immune receptors involved in recognizing RNA virus invasion. J. Virol. 86, 2900-2910. https://doi.org/10.1128/JVI.05738-11
  17. Kanda, T., Steele, R., Ray, R., and Ray, R.B. (2007). Hepatitis C virus infection induces the beta interferon signaling pathway in immortalized human hepatocytes. J. Virol. 81, 12375-12381. https://doi.org/10.1128/JVI.01695-07
  18. Kaneko, T., Tanji, Y., Satoh, S., Hijikata, M., Asabe, S., Kimura, K., and Shimotohno, K. (1994). Production of two phosphoproteins from the NS5A region of the hepatitis C viral genome. Biochem. Biophys. Res. Commun. 205, 320-326. https://doi.org/10.1006/bbrc.1994.2667
  19. Kawai, T. and Akira, S. (2006). Innate immune recognition of viral infection. Nat. Immunol. 7, 131-137. https://doi.org/10.1038/ni1303
  20. Kawai, T. and Akira, S. (2008). Toll-like receptor and RIG-I-like receptor signaling. Ann. N. Y. Acad. Sci. 1143, 1-20. https://doi.org/10.1196/annals.1443.020
  21. 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. https://doi.org/10.1038/ni1243
  22. Kumthip, K., Chusri, P., Jilg, N., Zhao, L., Fusco, D.N., Zhao, H., Goto, K., Cheng, D., Schaefer, E.A., Zhang, L., et al. (2012). Hepatitis C virus NS5A disrupts STAT1 phosphorylation and suppresses type I interferon signaling. J. Virol. 86, 8581-8591. https://doi.org/10.1128/JVI.00533-12
  23. Lan, K.H., Lan, K.L., Lee, W.P., Sheu, M.L., Chen, M.Y., Lee, Y.L., Yen, S.H., Chang, F.Y., and Lee, S.D. (2007). HCV NS5A inhibits interferon-alpha signaling through suppression of STAT1 phosphorylation in hepatocytederived cell lines. J. Hepatol. 46, 759-767.
  24. Lau, D.T., Fish, P.M., Sinha, M., Owen, D.M., Lemon, S.M., and Gale, M., Jr. (2008). Interferon regulatory factor-3 activation, hepatic interferonstimulated gene expression, and immune cell infiltration in hepatitis C virus patients. Hepatology 47, 799-809. https://doi.org/10.1002/hep.22076
  25. Li, K., Foy, E., Ferreon, J.C., Nakamura, M., Ferreon, A.C., Ikeda, M., Ray, S.C., Gale, M., Jr., and Lemon, S.M. (2005a). Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc. Natl. Acad. Sci. U. S. A. 102, 2992-2997. https://doi.org/10.1073/pnas.0408824102
  26. Li, Q., Brass, A.L., Ng, A., Hu, Z., Xavier, R.J., Liang, T.J., and Elledge, S.J. (2009). A genome-wide genetic screen for host factors required for hepatitis C virus propagation. Proc. Natl. Acad. Sci. U. S. A. 106, 16410-16415. https://doi.org/10.1073/pnas.0907439106
  27. Li, S., Wang, L., Berman, M., Kong, Y.Y., and Dorf, M.E. (2011). Mapping a dynamic innate immunity protein interaction network regulating type I interferon production. Immunity 35, 426-440. https://doi.org/10.1016/j.immuni.2011.06.014
  28. Li, X.D., Sun, L., Seth, R.B., Pineda, G., and Chen, Z.J. (2005b). Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity. Proc. Natl. Acad. Sci. U. S. A. 102, 17717-17722. https://doi.org/10.1073/pnas.0508531102
  29. Lim, Y.S., Nguyen, M.T.N., Pham, T.X., Huynh, T.T.X., Park, E.M., Choi, D.H., Kang, S.M., Tark, D., and Hwang, S.B. (2022). Hepatitis C virus NS5A protein interacts with telomere length regulation protein: implications for telomere shortening in patients infected with HCV. Mol. Cells 45, 148-157. https://doi.org/10.14348/molcells.2021.0167
  30. Lin, R., Heylbroeck, C., Pitha, P.M., and Hiscott, J. (1998). Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation. Mol. Cell. Biol. 18, 2986-2996. https://doi.org/10.1128/MCB.18.5.2986
  31. Lin, W., Kim, S.S., Yeung, E., Kamegaya, Y., Blackard, J.T., Kim, K.A., Holtzman, M.J., and Chung, R.T. (2006). Hepatitis C virus core protein blocks interferon signaling by interaction with the STAT1 SH2 domain. J. Virol. 80, 9226-9235. https://doi.org/10.1128/JVI.00459-06
  32. Lindenbach, B.D. and Rice, C.M. (2005). Unravelling hepatitis C virus replication from genome to function. Nature 436, 933-938. https://doi.org/10.1038/nature04077
  33. Masaki, T., Matsunaga, S., Takahashi, H., Nakashima, K., Kimura, Y., Ito, M., Matsuda, M., Murayama, A., Kato, T., Hirano, H., et al. (2014). Involvement of hepatitis C virus NS5A hyperphosphorylation mediated by casein kinase I-alpha in infectious virus production. J. Virol. 88, 7541-7555. https://doi.org/10.1128/JVI.03170-13
  34. Matsumoto, M., Hwang, S.B., Jeng, K.S., Zhu, N., and Lai, M.M. (1996). Homotypic interaction and multimerization of hepatitis C virus core protein. Virology 218, 43-51. https://doi.org/10.1006/viro.1996.0164
  35. Metz, P., Reuter, A., Bender, S., and Bartenschlager, R. (2013). Interferonstimulated genes and their role in controlling hepatitis C virus. J. Hepatol. 59, 1331-1341. https://doi.org/10.1016/j.jhep.2013.07.033
  36. Morikawa, K., Lange, C.M., Gouttenoire, J., Meylan, E., Brass, V., Penin, F., and Moradpour, D. (2011). Nonstructural protein 3-4A: the Swiss army knife of hepatitis C virus. J. Viral Hepat. 18, 305-315. https://doi.org/10.1111/j.1365-2893.2011.01451.x
  37. Nakatsu, Y., Matsuoka, M., Chang, T.H., Otsuki, N., Noda, M., Kimura, H., Sakai, K., Kato, H., Takeda, M., and Kubota, T. (2014). Functionally distinct effects of the C-terminal regions of IKKepsilon and TBK1 on type I IFN production. PLoS One 9, e94999. https://doi.org/10.1371/journal.pone.0094999
  38. Ng, S.L., Friedman, B.A., Schmid, S., Gertz, J., Myers, R.M., Tenoever, B.R., and Maniatis, T. (2011). IkappaB kinase epsilon (IKK(epsilon)) regulates the balance between type I and type II interferon responses. Proc. Natl. Acad. Sci. U. S. A. 108, 21170-21175. https://doi.org/10.1073/pnas.1119137109
  39. Nguyen, L.P., Nguyen, T.T.T., Nguyen, H.C., Pham, H.T., Han, K.M., Choi, D.H., Park, E.M., Kang, S.M., Tark, D., Lim, Y.S., et al. (2020). Cortactin interacts with hepatitis C virus core and NS5A proteins: implications for virion assembly. J. Virol. 94, e01306-20.
  40. Noguchi, T., Satoh, S., Noshi, T., Hatada, E., Fukuda, R., Kawai, A., Ikeda, S., Hijikata, M., and Shimotohno, K. (2001). Effects of mutation in hepatitis C virus nonstructural protein 5A on interferon resistance mediated by inhibition of PKR kinase activity in mammalian cells. Microbiol. Immunol. 45, 829-840. https://doi.org/10.1111/j.1348-0421.2001.tb01322.x
  41. Oshiumi, H., Sakai, K., Matsumoto, M., and Seya, T. (2010). DEAD/H BOX 3 (DDX3) helicase binds the RIG-I adaptor IPS-1 to up-regulate IFN-betainducing potential. Eur. J. Immunol. 40, 940-948. https://doi.org/10.1002/eji.200940203
  42. Park, K.J., Choi, S.H., Choi, D.H., Park, J.M., Yie, S.W., Lee, S.Y., and Hwang, S.B. (2003). Hepatitis C virus NS5A protein modulates c-Jun N-terminal kinase through interaction with tumor necrosis factor receptor-associated factor 2. J. Biol. Chem. 278, 30711-30718. https://doi.org/10.1074/jbc.M209623200
  43. Pavio, N., Taylor, D.R., and Lai, M.M. (2002). Detection of a novel unglycosylated form of hepatitis C virus E2 envelope protein that is located in the cytosol and interacts with PKR. J. Virol. 76, 1265-1272. https://doi.org/10.1128/JVI.76.3.1265-1272.2002
  44. Perwitasari, O., Cho, H., Diamond, M.S., and Gale, M., Jr. (2011). Inhibitor of kappaB kinase epsilon (IKK(epsilon)), STAT1, and IFIT2 proteins define novel innate immune effector pathway against West Nile virus infection. J. Biol. Chem. 286, 44412-44423. https://doi.org/10.1074/jbc.M111.285205
  45. Polyak, S.J., Khabar, K.S., Paschal, D.M., Ezelle, H.J., Duverlie, G., Barber, G.N., Levy, D.E., Mukaida, N., and Gretch, D.R. (2001). Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response. J. Virol. 75, 6095-6106. https://doi.org/10.1128/JVI.75.13.6095-6106.2001
  46. Ray, R.B. and Ray, R. (2001). Hepatitis C virus core protein: intriguing properties and functional relevance. FEMS Microbiol. Lett. 202, 149-156. https://doi.org/10.1111/j.1574-6968.2001.tb10796.x
  47. Reed, K.E. and Rice, C.M. (2000). Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Curr. Top. Microbiol. Immunol. 242, 55-84.
  48. Reed, K.E., Xu, J., and Rice, C.M. (1997). Phosphorylation of the hepatitis C virus NS5A protein in vitro and in vivo: properties of the NS5A-associated kinase. J. Virol. 71, 7187-7197. https://doi.org/10.1128/jvi.71.10.7187-7197.1997
  49. Reyes, G.R. (2002). The nonstructural NS5A protein of hepatitis C virus: an expanding, multifunctional role in enhancing hepatitis C virus pathogenesis. J. Biomed. Sci. 9, 187-197. https://doi.org/10.1007/BF02256065
  50. Saito, I., Miyamura, T., Ohbayashi, A., Harada, H., Katayama, T., Kikuchi, S., Watanabe, Y., Koi, S., Onji, M., and Ohta, Y. (1990). Hepatitis C virus infection is associated with the development of hepatocellular carcinoma. Proc. Natl. Acad. Sci. U. S. A. 87, 6547-6549. https://doi.org/10.1073/pnas.87.17.6547
  51. Saito, T., Owen, D.M., Jiang, F., Marcotrigiano, J., and Gale, M., Jr. (2008). Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA. Nature 454, 523-527. https://doi.org/10.1038/nature07106
  52. Schroder, M., Baran, M., and Bowie, A.G. (2008). Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKepsilon-mediated IRF activation. EMBO J. 27, 2147-2157. https://doi.org/10.1038/emboj.2008.143
  53. Seth, R.B., Sun, L., Ea, C.K., and Chen, Z.J. (2005). Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell 122, 669-682. https://doi.org/10.1016/j.cell.2005.08.012
  54. Sharma, S., tenOever, B.R., Grandvaux, N., Zhou, G.P., Lin, R., and Hiscott, J. (2003). Triggering the interferon antiviral response through an IKK-related pathway. Science 300, 1148-1151. https://doi.org/10.1126/science.1081315
  55. Tanji, Y., Kaneko, T., Satoh, S., and Shimotohno, K. (1995). Phosphorylation of hepatitis C virus-encoded nonstructural protein NS5A. J. Virol. 69, 3980-3986. https://doi.org/10.1128/jvi.69.7.3980-3986.1995
  56. Tenoever, B.R., Ng, S.L., Chua, M.A., McWhirter, S.M., Garcia-Sastre, A., and Maniatis, T. (2007). Multiple functions of the IKK-related kinase IKKepsilon in interferon-mediated antiviral immunity. Science 315, 1274-1278. https://doi.org/10.1126/science.1136567
  57. Wong, A.H., Tam, N.W., Yang, Y.L., Cuddihy, A.R., Li, S., Kirchhoff, S., Hauser, H., Decker, T., and Koromilas, A.E. (1997). Physical association between STAT1 and the interferon-inducible protein kinase PKR and implications for interferon and double-stranded RNA signaling pathways. EMBO J. 16, 1291-1304. https://doi.org/10.1093/emboj/16.6.1291
  58. Yu, M. and Levine, S.J. (2011). Toll-like receptor, RIG-I-like receptors and the NLRP3 inflammasome: key modulators of innate immune responses to double-stranded RNA viruses. Cytokine Growth Factor Rev. 22, 63-72. https://doi.org/10.1016/j.cytogfr.2011.02.001
  59. Yu, S., Chen, J., Wu, M., Chen, H., Kato, N., and Yuan, Z. (2010). Hepatitis B virus polymerase inhibits RIG-I- and Toll-like receptor 3-mediated beta interferon induction in human hepatocytes through interference with interferon regulatory factor 3 activation and dampening of the interaction between TBK1/IKKepsilon and DDX3. J. Gen. Virol. 91, 2080-2090. https://doi.org/10.1099/vir.0.020552-0