Functions of NS3 in HCV RNA Replication

描述(申请人提供)：慢性丙型肝炎病毒(丙型肝炎病毒)感染是严重肝病的常见原因，包括肝硬变和肝细胞癌。世界上大约2%的人口感染了这种病毒，使其成为主要的健康威胁。在美国，干扰素-α和利巴韦林的联合治疗是目前获得批准的治疗这种疾病的唯一方法，可以从不到50%的感染最常见的丙型肝炎病毒的人中消除病毒。丙型肝炎病毒NS3编码RNA复制所需的几种重要酶活性，包括蛋白酶、解旋酶和NTPase。NS3作为蛋白水解酶对病毒复制起着至关重要的作用，是病毒复制酶复合体组装所必需的，因此也是抗病毒药物开发的重要靶点。本项目的目的是通过检测在瞬时复制水平上表现出显著差异的丙型肝炎病毒复制子突变体，了解NS3蛋白在培养的Huh7细胞中作用于丙型肝炎病毒RNA复制的分子机制。本项目的目的是：特定的目的1：确定NS3的复制增强突变是否改变了丙型肝炎病毒非结构蛋白的处理，并确定改变的非结构蛋白处理是否导致差异RNA复制；特定的目的2：确定NS3是否通过与NS5A的相互作用来调节RNA的复制。这一应用的结果将增强有关病毒RNA复制的基本分子生物学知识，改进目前可用的研究丙型肝炎病毒的系统，并有助于未来的药物开发努力，从而加强对这种危险病原体的治疗。

DESCRIPTION (provided by applicant): Chronic hepatitis C virus (HCV) infection is a common cause of serious liver diseases, including cirrhosis and hepatocellular carcinoma. Approximately 2% of the world's population is infected with this virus, rendering it a major health threat. In the U.S., treatment with a combination of interferon-alpha and ribavirin, the only currently licensed therapies for this disease, eliminates the virus from less than 50% of those infected with the most prevalent HCV genotype, genotype 1. HCV NS3 encodes several important enzyme activities required for RNA replication, including protease, helicase and NTPase. NS3 as the protease is essential for viral replication as it proteolytically processes viral polyprotein, which is required for viral replicase complex assembly, and thus an important target in antiviral drug development efforts against HCV infection. The goal of this project is to understand the molecular mechanism of action of NS3 protein in HCV RNA replication in cultured Huh7 cells, by examining HCV replicon mutants, which have shown significant differences in transient replication levels, with a few amino acid changes within the protease domain of the NS3. The aims of this project are: Specific aim1: To determine if replication-enhancing mutations in NS3 alter HCV nonstructural protein processing and to determine whether altered nonstructural protein processing results in differential RNA replication; Specific aim 2: To determine if NS3 modulates RNA replication through interaction with NS5A. The outcome of this application will enhance basic molecular biological knowledge concerning viral RNA replication, improve the currently available system to study HCV, and help future drug development efforts leading to enhanced therapy against this dangerous pathogen.