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

描述（由申请人提供）：HCV感染持续存在的机制尚不清楚。HCV E2和NS5A可抑制细胞抗病毒反应；然而，数据一直存在争议。在与Michael Gale博士的团队合作中，我们最近证明了NS3/4A丝氨酸蛋白酶阻断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，这种阻断的详细后果，即下游肝细胞抗病毒防御基因表达的改变，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蛋白酶阻断IRF-3磷酸化的机制尚不清楚。本提案的主要目标是利用最近发展的功能基因组学方法，包括微阵列和蛋白质组学，探索这些问题的答案。特异性目的1，鉴定和分析被NS3/4A表达或基因组长度HCV RNA复制阻断/抑制的肝细胞中IRF-3依赖和独立的抗病毒应答基因。(1)有条件地表达NS3/4A蛋白酶，(2)含有复制的HCV RNA，或(3)有条件地表达显性阴性IRF-3突变体的细胞将受到仙台病毒（SenV）的攻击，使用Affymetrix微阵列鉴定SenV激活基因表达的差异，并与克隆匹配的HCV阴性细胞进行比较。这些实验应该确定IRF-3通路下游的抗病毒基因，以及其他可能被NS3/4A蛋白酶阻断/抑制的通路。它们还将提供其他HCV蛋白和IRF-3独立通路是否有助于HCV感染的持续存在的信息。特异性目的2，利用蛋白质组学方法探索NS3/4A蛋白酶阻断IRF-3磷酸化在蛋白水平上的细胞变化。将条件表达NS3/4A蛋白酶的细胞用SenV激射，通过二维凝胶电泳结合自动多肽取样和质谱多肽测序，鉴定激射前后、存在和不存在NS3/4A的蛋白表达差异。这种方法很可能鉴定出病毒激活激酶（VAK）或其他被NS3/4A丝氨酸蛋白酶抑制的病毒诱导的IRF-3磷酸化所必需的信号成分。这一建议将有助于更好地理解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.