Functions of NS3 in HCV RNA Replication

描述（由申请人提供）：慢性丙型肝炎病毒（HCV）感染是严重肝脏疾病的常见原因，包括肝硬化和肝细胞癌。世界上大约2%的人口感染了这种病毒，使其成为一个重大的健康威胁。在美国，干扰素- α和利巴韦林联合治疗是目前唯一获得许可的治疗这种疾病的方法，可以从最流行的HCV基因型（基因1型）感染者中消除不到50%的病毒。HCV 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作为一种蛋白酶对病毒复制至关重要，因为它对病毒多蛋白进行蛋白水解处理，而病毒多蛋白是病毒复制酶复合体组装所必需的，因此是开发抗HCV感染抗病毒药物的重要靶点。本项目的目的是通过检测HCV复制子突变体，了解NS3蛋白在培养的Huh7细胞中HCV RNA复制中的分子机制，这些突变体在瞬时复制水平上表现出显著差异，在NS3蛋白酶结构域内发生了一些氨基酸变化。该项目的目的是：特异性目的1：确定NS3复制增强突变是否改变HCV非结构蛋白加工，并确定非结构蛋白加工的改变是否导致差异RNA复制；特异性目的2：确定NS3是否通过与NS5A的相互作用调节RNA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HCV研究系统，并有助于未来的药物开发工作，从而增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.