Presenter: Dr. Chunfu Zheng

University: Wuhan Institute of Virology, Chinese Academy of Sciences

Time: 9:30-10:30, December 12, 2011

Venue: A202, Institute of Microbiology, Chinese Academy of Sciences

Abstract: 1. Varicella-zoster virus (VZV) infection of differentiated cells within the host and establishment of latency likely requires evasion of innate immunity and limits secretion of antiviral cytokines. Here we report that its immediate-early protein ORF61 antagonizes the beta interferon (IFN-beta) pathway. VZV infection down-modulated the Sendai virus (SeV)-activated IFN-beta pathway, including mRNA of IFN-beta and its downstream interferon-stimulated genes (ISGs), ISG54 and ISG56. Through a primary screening of VZV genes, we found that ORF61 inhibited SeV-mediated activation of IFN-betaand ISRE (IFN-stimulated response element) promoter activities but only slightly affected NF-kB promoter activity, implying that the IFN-beta pathway may be blocked in the IRF3 branch. An indirect immunofluorescence assay demonstrated that ectopic expression of ORF61 abrogated the detection of IRF3 in SeV-infected cells; however, it did not affect endogenous dormant IRF3 in noninfected cells. Additionally, ORF61 was shown to be partially colocalized with activated IRF3 in the nucleus upon treatment with MG132, an inhibitor of proteasomes, and the direct interaction between ORF61 and activated IRF3 was confirmed by a coimmunoprecipitation assay. Furthermore, Western blot analysis demonstrated that activated IRF3 was ubiquitinated in the presence of ORF61, suggesting that ORF61 degraded phosphorylated IRF3 via a ubiquitin-proteasome pathway. Semiquantitative reverse transcription-PCR (RT-PCR) analysis demonstrated that the level of ISG54 and ISG56 mRNAs was also downregulated by ORF61. Taken together, our results convincingly demonstrate that ORF61 down-modulates the IRF3-mediated IFN-beta pathway by degradation of activated IRF3 via direct interaction, which may contribute to the pathogenesis of VZV infection.

2. The interferon (IFN)-mediated antiviral response is the major defense of the host immune system. In order to complete their life cycle, viruses must modulate host IFN-mediated immune responses. Herpes simplex virus 1 (HSV-1) is a large DNA virus containing over 80 genes, many of which encode proteins that are involved in virus-host interactions and show immune modulatory capabilities. In this study, we demonstrate that the US11 protein, an RNA binding tegument protein of HSV-1, is a novel antagonism of the interferon　(IFN- ) pathway. US11 significantly inhibited the Sendai virus (SeV)-mediated activation of the IFN- promoter, and its dsRNA binding domain was indispensable for this inhibition activity. However, US11 could not inhibit IFN- promoter activity induced by overexpression of the N-terminal CARD domain of RIG-I or MDA-5 and their downstream signaling molecules including IPS-1/MAVS, TBK1, IKKi and IRF3, but could inhibit IFN- promoter activity induced by overexpression of the full-length MDA-5, indicating that US11 may block the IFN- pathway at the RIG-I and MDA-5 level. Co-immunoprecipitation analysis demonstrated that US11 could interact with both RIG-I and MDA-5 through its C-terminal RNA-binding domain, which was RNA-independent. Expression of US11 in both transfected and HSV-1 infected cells could interfere with the interaction between MAVS and RIG-I or MDA-5. Furthermore, US11 was shown to dampen SeV-mediated IRF3 activation. Taken together, the combined data indicate that HSV-1 US11 binds to RIG-I and MDA-5 and inhibits their downstream signaling pathway, preventing the transcriptional induction of IFN- , which may contribute to the pathogenesis of HSV-1 infection.