Abstract
Chronic hepatitis C virus (HCV) infection is a major global public health problem. HCV infection is supported by viral strategies to evade the innate antiviral response wherein the viral NS3.4A protease complex targets and cleaves the interferon promoter stimulator-1 (IPS-1) adaptor protein to ablate signaling of interferon alpha/beta immune defenses. Here we examined the structural requirements of NS3.4A and the therapeutic potential of NS3.4A inhibitors to control the innate immune response against virus infection. The structural composition of NS3 includes an amino-terminal serine protease domain and a carboxyl-terminal RNA helicase domain. NS3 mutants lacking the helicase domain retained the ability to control virus signaling initiated by retinoic acid-inducible gene-I (RIG-I) or melanoma differentiation antigen 5 and suppressed the downstream activation of interferon regulatory factor-3 (IRF-3) and nuclear factor kappaB (NF-kappaB) through the targeted proteolysis of IPS-1. This regulation was abrogated by truncation of the NS3 protease domain or by point mutations that ablated protease activity. NS3.4A protease control of antiviral immune signaling was due to targeted proteolysis of IPS-1 by the NS3 protease domain and minimal NS4A cofactor. Treatment of HCV-infected cells with an NS3 protease inhibitor prevented IPS-1 proteolysis by the HCV protease and restored RIG-I immune defense signaling during infection. Thus, the NS3.4A protease domain can target IPS-1 for cleavage and is essential for blocking RIG-I signaling to IRF-3 and NF-kappaB, whereas the helicase domain is dispensable for this action. Our results indicate that NS3.4A protease inhibitors have immunomodulatory potential to restore innate immune defenses to HCV infection.
Highlights
10792 JOURNAL OF BIOLOGICAL CHEMISTRY lion people worldwide with chronic hepatitis C virus (HCV) infection, and it is a major cause of hepatitis and liver disease
Our results show that the NS3 protease domain and minimal NS4A cofactor are sufficient to inhibit antiviral signaling through Retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) and block activation of the downstream transcription factors IFN regulatory factor-3 (IRF-3) and nuclear factor-B (NF-B)
Infection studies demonstrated for the first time that NS31⁄74A protease inhibitor therapy of HCV infection can effectively remove the NS31⁄74A blockade to the innate immune response, restoring RIG-I signaling to IFN- promoter stimulator 1 (IPS-1) and activation of IFN-stimulated gene (ISG) expression in infected cells
Summary
Expression Cloning and Site-directed Mutagenesis—pFLAG NS31⁄74A and pFLAG NS3 were generated as described [23]. The NS31⁄74A blockade of NF-B binding was relieved when cells were treated with SCH6 or ITMN-C peptidomimetic active site inhibitors of the NS31⁄74A protease (lanes 13 and 15, respectively) These results were verified by luciferase reporter assay, in which WT NS31⁄74A suppressed Sendai virus induction of an NF-B-dependent PRDII-luciferase promoter and its enhancement by ectopic RIG-I or MDA5 (Fig. 6B). To determine the domain of NS31⁄74A necessary for this effect, HEK 293 cells were transiently transfected with the indicated NS3 construct followed by Sendai virus infection (Fig. 6C, left panel) or were cotransfected with a construct encoding constitutively active N-RIG to trigger PRDII promoter signaling (Fig. 6C, right panel). ITMN-C Restores the Endogenous Innate Immune Response Triggered by HCV RNA Replication and HCV Infection—To examine the effect of protease inhibitor treatment on IPS-1 and the innate antiviral response against HCV, we examined ITMN-C treatment of Huh cells harboring a HCV 2a replicon
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