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

描述（由申请人提供）：对HCV感染持续存在的机制知之甚少。HCV E2和NS 5A被认为可以抑制细胞的抗病毒反应;然而，数据一直存在争议。在与Michael Gale博士的研究小组合作中，我们最近证明了NS 3/4A丝氨酸蛋白酶阻断了病毒诱导的磷酸化和干扰素调节因子3（IRF-3）的激活，IRF-3是启动细胞抗病毒反应的关键转录因子。然而，这种阻断的详细后果，即下游肝细胞抗病毒防御基因表达的改变，这可能有助于丙型肝炎的持续存在，仍有待完全阐明。此外，NS 3/4A蛋白酶阻断IRF-3磷酸化的机制尚不清楚。本研究的主要目的是利用近年来发展起来的功能基因组学方法，包括微阵列和蛋白质组学，来探索这些问题的答案。具体目的1，鉴定和分析肝细胞中被NS 3/4A表达或基因组长度HCV RNA复制阻断/抑制的IRF-3依赖性和独立性抗病毒应答基因。将用仙台病毒（SenV）攻击（1）条件性表达NS 3/4A蛋白酶、（2）含有复制型全长HCV RNA或（3）条件性表达显性阴性IRF-3突变体的细胞，并使用Affytron微阵列鉴定SenV激活基因表达的差异，并与来自克隆匹配的HCV阴性细胞的差异进行比较。这些实验应该确定IRF-3途径下游的抗病毒基因和其他可能被NS 3/4A蛋白酶阻断/抑制的途径。他们还将提供其他HCV蛋白和IRF-3独立途径是否有助于HCV感染持续存在的信息。具体目的2，利用蛋白质组学方法，探讨NS 3/4A蛋白酶阻断IRF-3磷酸化的蛋白质水平的细胞变化。条件性表达NS 3/4A蛋白酶的细胞将用SenV攻击，并且在存在和不存在NS 3/4A的情况下，攻击前后蛋白质表达的差异将通过二维凝胶电泳结合自动多肽取样和通过质谱法进行多肽测序来鉴定。这种方法有可能确定病毒激活的激酶（VAK）或其他信号传导组件不可或缺的病毒诱导的IRF-3磷酸化，抑制NS 3/4A丝氨酸蛋白酶。这一建议将导致更好地了解HCV如何破坏先天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.