MECHANISMS OF HEPATITIS C VIRUS RNA REPLICATION

丙型肝炎病毒(丙型肝炎病毒)是一种新近发现的包膜单细胞病毒。 滞留的RNA病毒已被证明是引起 输血后和零星获得性非甲非乙型肝炎。一半 在所有的丙型肝炎病毒感染中，20%的人发展为慢性肝炎 会导致肝硬变。慢性丙型肝炎病毒感染也与 肝细胞癌的发展。目前对该问题的理解 这种病毒的生命周期在很大程度上是基于其基因组的推断 序列，所推导的单个编码多蛋白的结构，以及 从其他病毒系统获得的知识。这个项目的目标是 为了扩大我们对丙型肝炎病毒复制机制的理解， 特别关注病毒复制酶的活性， 模板RNA的结构要求和宿主细胞的作用 各种因素。序列分析表明，丙型肝炎病毒多聚蛋白含有 RNA聚合酶、RNA解旋酶和 丝氨酸蛋白酶。在初步调查中，我们已经确认和 从提取物中部分纯化依赖RNA的RNA聚合酶活性 受感染的人类肝细胞。这项活动，没有出现在 未感染细胞提取物，能够通过以下方式复制丙型肝炎病毒RNA序列 延伸与模板RNA杂交的合成引物。我们计划 鉴定这种丙型肝炎病毒特异性聚合酶的酶活性， 包括其产品、底物专一性、辅因子要求以及 对竞争性和非竞争性抑制剂的敏感性。在……里面 此外，我们将使用存在的丙型肝炎病毒聚合酶活性来鉴定 体内丙型肝炎病毒肝外复制部位、细胞培养鉴定 适合病毒复制的体外模型的系统，以及 细胞内RNA复制复合体的特征。这些研究 应有助于识别其他病毒编码的和细胞RNA 复制复合体。这些研究应该有助于识别 支持和调节的其他病毒编码和细胞蛋白 病毒RNA复制，包括负责RNA序列的活动 特异性、RNA合成的启动和复制的保真度。在……里面 单独的研究，我们将鉴定，鉴定和纯化丙型肝炎病毒- 特定的RNA解旋酶，一种可能促进RNA复制的酶 通过催化RNA双链结构的解旋。我们会 表征动力学、底物专一性和辅因子 这种酶的要求，以区别于各种 从这些研究中获得的解旋酶将有助于设计和测试 一系列新型化合物，有效地、选择性地抑制 这种病毒的复制。总体而言，通过扩大我们对 病毒复制的分子机制，这些研究将加强 我们能够中断丙型肝炎病毒的生命周期并限制通常严重的 丙型肝炎病毒感染的临床表现。

Hepatitis C virus (HCV) is a recently-identified, enveloped, single- stranded RNA virus that has been shown to be the predominant cause of post-transfusion and sporadically-acquired non-A, non-B hepatitis. Half of all HCV infections progress to chronic hepatitis, and 20% of these lead to cirrhosis. Chronic HCV infection is also associated with the development of hepatocellular carcinoma. Current understanding of the lifecycle of this virus is based largely on inference from its genomic sequence, the deduced structure of the single encoded polyprotein, and knowledge gained from other viral systems. The aims of this project are to extend our understanding of the mechanisms of HCV replication, with particular focus on the activities of viral replicative enzymes, the structural requirements of template RNA, and the role of host cellular factors. Sequence analysis reveals that the HCV polyprotein contains separate structural motifs common to RNA polymerases, RNA helicases, and serine proteases. In preliminary investigations, we have identified and partially purified an RNA-dependent RNA polymerase activity from extracts of infected human hepatocytes. This activity, which is absent from uninfected cell extracts, is capable of replicating HCV RNA sequences by extending a synthetic primer hybridized to the template RNA. We plan to characterize the enzymatic activities of this HCV-specific polymerase, including its products, substrate specificity, cofactor requirements, and susceptibility to competitive and non-competitive inhibitors. In addition, we will use the presence of HCV polymerase activity to identify extrahepatic sites of HCV replication in vivo, to identify cell culture systems as suitable in vitro models of viral replication, and to characterize intracellular RNA replication complexes. These studies should facilitate identification of other virus-encoded and cellular RNA replication complexes. These studies should facilitate identification of other virus-encoded and cellular proteins that support and regulate viral RNA replication, including activities responsible for RNA sequence specificity, initiation of RNA synthesis, and copying fidelity. In separate studies, we will identify, characterize and purify the HCV- specific RNA helicase, an enzyme that likely facilitates RNA replication by catalyzing the unwinding of RNA duplex structures. We will characterize the kinetics, substrate specificity, and cofactor requirements of this enzyme to distinguish it from the variety of helicase gained from these studies will facilitate the design and testing of novel compounds that potently, and selectively, inhibit the replication of this virus. Overall, by extending our understanding of the molecular mechanisms of viral replication, these studies will enhance our ability to interrupt the HCV lifecycle and limit the often severe clinical manifestations of HCV infection.