Impact of HCV NS3/4A Protease on Host Innate Immunity

描述(由申请人提供)：丙型肝炎病毒感染持续存在的机制尚不清楚。丙型肝炎病毒E2和NS5A被认为可以抑制细胞抗病毒反应；然而，数据一直存在争议。最近，我们与Michael Gale博士的团队合作，证明了NS3/4A丝氨酸蛋白酶可以阻止病毒诱导的干扰素调节因子3(IRF-3)的磷酸化和激活，IRF-3是启动细胞抗病毒反应的关键转录因子。然而，这种阻断的具体后果，即下游肝细胞抗病毒防御基因表达的变化，可能有助于丙型肝炎的持续存在，仍有待完全阐明。此外，NS3/4A蛋白酶阻止IRF-3磷酸化的机制尚不清楚。这项提议的主要目标是使用最近开发的功能基因组学方法来探索这些问题的答案，包括微阵列和蛋白质组学。具体目的1，鉴定和分析被NS3/4A表达或基因组长度的丙型肝炎病毒RNA复制阻断/抑制的IRF-3依赖和独立的肝细胞抗病毒反应基因。(1)有条件地表达NS3/4A蛋白酶，(2)含有复制的全长丙型肝炎病毒RNA，或(3)有条件表达显性阴性IRF-3突变体的细胞将受到仙台病毒(SenV)的攻击，SenV激活的基因表达的差异将使用Affymetrix微阵列进行鉴定，并与克隆匹配的丙型肝炎病毒阴性细胞的差异进行比较。这些实验应该识别IRF-3途径下游的抗病毒基因，以及其他被NS3/4A蛋白酶阻断/抑制的可能途径。他们还将提供有关其他丙型肝炎病毒蛋白和IRF-3独立途径是否有助于丙型肝炎病毒感染持续的信息。具体目的2，用蛋白质组学方法研究NS3/4A蛋白水解酶阻断IRF-3磷酸化的细胞内蛋白水平的变化。有条件表达NS3/4A蛋白水解酶的细胞将被SenV攻击，并且在有或没有NS3/4A的情况下，攻击前后的蛋白质表达的差异将通过双向凝胶电泳法结合自动多肽采样和质谱仪多肽测序来鉴定。这种方法很可能识别病毒激活的激酶(VAK)或其他信号成分，这些信号成分对于病毒诱导的IRF-3磷酸化是必不可少的，这些信号成分被NS3/4A丝氨酸蛋白酶抑制。这一建议将有助于更好地理解丙型肝炎病毒如何扰乱天然免疫并导致肝细胞持续感染的机制。

DESCRIPTION (provided by applicant): The mechanisms underlying the persistence of HCV infection are poorly understood. HCV E2 and NS5A have been suggested to inhibit cellular antiviral responses; however, data have been controversial. In collaboration with Dr. Michael Gale's group, we recently demonstrated that the NS3/4A serine protease blocks virus-induced phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a key transcriptional factor in initiating cellular antiviral responses. However, the detailed consequences of this blockade, i.e. the alterations of downstream hepatocellular antiviral defensive gene expression, which may contribute to the persistence of hepatitis C, remain to be completely elucidated. In addition, the mechanisms by which NS3/4A protease blocks IRF-3 phosphorylation are yet not known. The primary goal of this proposal is to explore the answers of these issues using the recently-developed functional genomics approaches including microarrays and proteomics. Specific Aim 1, to identify and profile IRF-3 dependent and independent antiviral response genes of hepatocytes that are blocked /suppressed by NS3/4A expression or by genome-length HCV RNA replication. Cells that (1) conditionally express the NS3/4A protease, (2) contain replicating full-length HCV RNA, or (3) conditionally express a dominant negative IRF-3 mutant will be challenged with Sendai virus (SenV), and differences in SenV-activated gene expression will be identified using Affymetrix microarrays and compared with those from clonally matched HCV-negative cells. These experiments should identify antiviral genes downstream of the IRF-3 pathway and other possible pathways that are blocked/suppressed by NS3/4A protease. They will also provide information on whether other HCV proteins and IRF-3 independent pathways contribute to the persistence of HCV infection. Specific Aim 2, to explore the cellular changes at protein level by which NS3/4A protease blocks IRF-3 phosphorylation using a proteomics approach. Cells that conditionally express the NS3/4A protease will be challenged with SenV, and differences in protein expression before and after challenge, in the presence and absence of NS3/4A will be identified by two-dimensional gel eletrophoresis coupled with automated polypeptide sampling and polypeptide sequencing by mass spectrometry. This approach is likely to identify the virus-activated kinase (VAK) or other signaling components indispensable for virus-induced IRF-3 phosphorylation that are inhibited by the NS3/4A serine protease. This proposal should lead to a better understanding of the mechanisms how HCV disrupts the innate immunity and causes persistent infection in hepatocytes.