Hepatitis C virus NS4B protein targets STING and abrogates RIG-I–mediated type I interferon-dependent innate immunity§

Authors

  • Sayuri Nitta,

    1. Departments of Gastroenterology and HepatologyTokyo Medical and Dental University, Tokyo, Japan
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  • Naoya Sakamoto,

    Corresponding author
    1. Departments of Gastroenterology and HepatologyTokyo Medical and Dental University, Tokyo, Japan
    2. Departments of Hepatitis Control, Tokyo Medical and Dental University, Tokyo, Japan
    3. Department of Gastroenterology and Hepatology, Hokkaido University, Hokkaido, Japan
    • Department of Gastroenterology and Hepatology, Hokkaido University, Kita15, Nishi8, Kita-ku, Sapporo, Hokkaido, 060-0808, Japan
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    • These authors contributed equally to this work.

    • fax (81)-11-706-8036

  • Mina Nakagawa,

    1. Departments of Gastroenterology and HepatologyTokyo Medical and Dental University, Tokyo, Japan
    2. Departments of Hepatitis Control, Tokyo Medical and Dental University, Tokyo, Japan
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  • Sei Kakinuma,

    1. Departments of Gastroenterology and HepatologyTokyo Medical and Dental University, Tokyo, Japan
    2. Departments of Hepatitis Control, Tokyo Medical and Dental University, Tokyo, Japan
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  • Kako Mishima,

    1. Departments of Gastroenterology and HepatologyTokyo Medical and Dental University, Tokyo, Japan
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  • Akiko Kusano-Kitazume,

    1. Departments of Gastroenterology and HepatologyTokyo Medical and Dental University, Tokyo, Japan
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  • Kei Kiyohashi,

    1. Departments of Gastroenterology and HepatologyTokyo Medical and Dental University, Tokyo, Japan
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  • Miyako Murakawa,

    1. Departments of Gastroenterology and HepatologyTokyo Medical and Dental University, Tokyo, Japan
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  • Yuki Nishimura-Sakurai,

    1. Departments of Gastroenterology and HepatologyTokyo Medical and Dental University, Tokyo, Japan
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  • Seishin Azuma,

    1. Departments of Gastroenterology and HepatologyTokyo Medical and Dental University, Tokyo, Japan
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  • Megumi Tasaka-Fujita,

    1. Departments of Gastroenterology and HepatologyTokyo Medical and Dental University, Tokyo, Japan
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  • Yasuhiro Asahina,

    1. Departments of Gastroenterology and HepatologyTokyo Medical and Dental University, Tokyo, Japan
    2. Departments of Hepatitis Control, Tokyo Medical and Dental University, Tokyo, Japan
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  • Mitsutoshi Yoneyama,

    1. Division of Molecular Immunology, Medical Mycology Research Center, Chiba University, Chiba, Japan
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  • Takashi Fujita,

    1. Laboratory of Molecular Genetics, Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Kyoto, Japan
    2. Laboratory of Molecular Cell Biology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
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  • Mamoru Watanabe

    1. Departments of Gastroenterology and HepatologyTokyo Medical and Dental University, Tokyo, Japan
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  • Potential conflict of interest: Nothing to report.

  • BiFC, bimolecular fluorescence complementation; CARD, caspase recruitment domain; DAPI, 4',6-diamidino-2-phenylindole; dsRNA, double-stranded RNA; ER, endoplasmic reticulum; FACL4, fatty acid-CoA ligase, long chain 4; HCV, hepatitis C virus; IFN, interferon; IKKϵ, IκB kinase ϵ; IRF-3, interferon-regulatory factor 3; ISRE, interferon-stimulated response element; MAM, mitochondria-associated ER membrane; mKG, monomeric Kusabira-Green; PDI, protein disulphide-isomerase; pIRF-3, phosphorylated IRF3; poly(dA:dT), poly(deoxyadenylic-deoxythymidylic) acid; RIG-I, retinoic acid–inducible gene I; siRNA, small interfering RNA; SOCS, suppressor of cytokine signaling; STAT1, signal transducer and activator of transcription protein-1; STING, stimulator of interferon genes; TBK1, TANK binding kinase 1.

Abstract

Hepatitis C virus (HCV) infection blocks cellular interferon (IFN)-mediated antiviral signaling through cleavage of Cardif by HCV-NS3/4A serine protease. Like NS3/4A, NS4B protein strongly blocks IFN-β production signaling mediated by retinoic acid–inducible gene I (RIG-I); however, the underlying molecular mechanisms are not well understood. Recently, the stimulator of interferon genes (STING) was identified as an activator of RIG-I signaling. STING possesses a structural homology domain with flaviviral NS4B, which suggests a direct protein-protein interaction. In the present study, we investigated the molecular mechanisms by which NS4B targets RIG-I–induced and STING-mediated IFN-β production signaling. IFN-β promoter reporter assay showed that IFN-β promoter activation induced by RIG-I or Cardif was significantly suppressed by both NS4B and NS3/4A, whereas STING-induced IFN-β activation was suppressed by NS4B but not by NS3/4A, suggesting that NS4B had a distinct point of interaction. Immunostaining showed that STING colocalized with NS4B in the endoplasmic reticulum. Immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays demonstrated that NS4B specifically bound STING. Intriguingly, NS4B expression blocked the protein interaction between STING and Cardif, which is required for robust IFN-β activation. NS4B truncation assays showed that its N terminus, containing the STING homology domain, was necessary for the suppression of IFN-β promoter activation. NS4B suppressed residual IFN-β activation by an NS3/4A-cleaved Cardif (Cardif1-508), suggesting that NS3/4A and NS4B may cooperate in the blockade of IFN-β production. Conclusion: NS4B suppresses RIG-I–mediated IFN-β production signaling through a direct protein interaction with STING. Disruption of that interaction may restore cellular antiviral responses and may constitute a novel therapeutic strategy for the eradication of HCV. (HEPATOLOGY 2013)

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