Hepatitis C virus NS2 protease inhibits host cell antiviral response by inhibiting IKKε and TBK1 functions.

Hepatitis C virus (HCV) encodes for several proteins that can interfere with host cell signaling and antiviral response. Previously, serine protease NS3/4A was shown to block host cell interferon (IFN) production by proteolytic cleavage of MAVS and TRIF, the adaptor molecules of the RIG-I and TLR3 signaling pathways, respectively. This study shows that another HCV protease, NS2 can interfere efficiently with cytokine gene expression. NS2 and its proteolytically inactive mutant forms were able to inhibit type I and type III IFN, CCL5 and CXCL10 gene promoters activated by Sendai virus infection. However, the CXCL8 gene promoter was not inhibited by NS2. In addition, constitutively active RIG-I (ΔRIG-I), MAVS, TRIF, IKKε, and TBK1-induced activation of IFN-ß promoter was inhibited by NS2. Cotransfection experiments with IKKε or TBK1 together with interferon regulatory factor 3 (IRF3) and HCV expression constructs revealed that NS2 in a dose-dependent manner inhibited IKKε and especially TBK1-induced IRF3 phosphorylation. GST pull-down experiments with GST-NS2 and in vitro-translated and cell-expressed IKKε and TBK1 demonstrated direct physical interactions of the kinases with NS2. Further evidence that the IKKε/TBK1 kinase complex is the target for NS2 was obtained from the observation that the constitutively active form of IRF3 (IRF3-5D) activated readily IFN-ß promoter in the presence of NS2. The present study identified HCV NS2 as a potent interferon antagonist, and describes an explanation of how NS2 downregulates the major signaling pathways involved in the development of host innate antiviral responses.

Human kinome analysis reveals novel kinases contributing to virus infection and retinoic-acid inducible gene I-induced type I and type III IFN gene expression.

Activation of host innate antiviral responses are mediated by retinoic-acid inducible gene I (RIG-I)-like receptors, RIG-I and melanoma differentiation-associated gene 5, and TLRs 3, 7, 8 and 9, recognising different types of viral nucleic acids. The major components of the RIG-I- and TLR pathways have putatively been identified, but previously unrecognised kinases may contribute to virus infection-induced activation of the IFN response. Here, we screened a human kinase cDNA library, termed the kinome, using an IFN-λ1 promoter-driven luciferase reporter assay in HEK293 cells during Sendai virus infection. Of the 568 kinases analysed, nearly 50 enhanced IFN-λ1 gene expression at least twofold in response to Sendai virus infection. The best activators were FYN (FYN oncogene related to SRC, FGR, YES), serine/threonine kinase 24, activin A receptor type 1 and SRPK1 (SFRS protein kinase 1). These kinases enhanced RIG-I-dependent IFN-λ1 promoter activation via IFN-stimulated response and NF-κB elements, as confirmed using mutant IFN-λ1 promoter constructs. FYN and SRPK1 enhanced IFN-λ1 and CXCL10 protein production via the RIG-I pathway, and stimulated RIG-I and MyD88-dependent phosphorylation of IRF3 and IRF7 transcription factors, respectively. We conclude that several previously unrecognised kinases, particularly FYN and SRPK1, positively regulate IFN-λ1 and similarly regulated cytokine and chemokine genes during viral infection.