MECHANISMS OF HEPATITIS C VIRUS RNA REPLICATION

丙型肝炎病毒（HCV）是一种新近发现的有包膜的单- 链RNA病毒已被证明是主要原因， 输血后和偶发性非甲非乙型肝炎。 一半 所有HCV感染进展为慢性肝炎，其中20% 导致肝硬化。 慢性HCV感染也与 肝细胞癌的发展。 目前了解的 这种病毒的生命周期很大程度上是基于其基因组推断， 序列，单一编码多蛋白的推导结构，和 从其他病毒系统中获得的知识。 该项目的目标是 为了扩大我们对HCV复制机制的理解， 特别关注病毒复制酶的活动， 模板RNA的结构要求，以及宿主细胞的作用 因素 序列分析表明，HCV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，我们发现， 从提取物中部分纯化RNA依赖性RNA聚合酶活性 感染的人类肝细胞。 这种活动，这是缺席 未感染的细胞提取物，能够复制HCV RNA序列， 延伸与所述模板RNA杂交的合成引物。 我们计划 表征这种HCV特异性聚合酶的酶活性， 包括其产物、底物特异性、辅因子要求，以及 对竞争性和非竞争性抑制剂的敏感性。 在 此外，我们将使用HCV聚合酶活性的存在来鉴定 HCV在体内复制的肝外位点，以鉴定细胞培养物 系统作为病毒复制的合适体外模型，以及 表征细胞内RNA复制复合物。 这些研究 应有助于鉴定其他病毒编码和细胞RNA 复制复合体 这些研究应有助于查明 其他病毒编码的和细胞蛋白质， 病毒RNA复制，包括负责RNA序列的活动 特异性、RNA合成起始和复制保真度。 在 单独的研究，我们将识别，表征和纯化HCV- 特异性RNA解旋酶，可能促进RNA复制的酶 通过催化RNA双链体结构的解旋。 我们将 表征动力学、底物特异性和辅因子 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 新的化合物，有效地，选择性地抑制， 这种病毒的复制。 总的来说，通过扩展我们对 病毒复制的分子机制，这些研究将提高 我们中断HCV生命周期并限制通常严重的 HCV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.