Whole-genome siRNA screen for host genes supporting hepatitis C virus replication

项目概述：慢性丙型肝炎病毒（HCV）感染影响了世界上约3%的人口，HCV肝硬化现在是美国肝移植的主要指征。目前治疗慢性丙型肝炎病毒感染的最佳方法（聚乙二醇干扰素和利巴韦林联合治疗）只能在大约一半的治疗个体中产生持续的病毒学反应。然而，有一些亚群体的治疗反应率要低得多，比如非裔美国人和艾滋病合并感染者。因此，迫切需要新的丙型肝炎病毒治疗方法。HCV蛋白的小分子抑制剂很有前景，但很明显，病毒对这类药物的耐药性发展很快。我们建议鉴定作为病毒复制辅助因子的宿主基因，理由是细胞辅助因子可能是HCV治疗的靶点。例如，亲环蛋白B是一种宿主蛋白，已被证明参与HCV复制，亲环蛋白B的小分子抑制剂在体外和体内都是HCV复制的有效抑制剂。在这个应用程序中，我们将筛选整个人类基因组siRNA文库针对HCV复制子细胞系编码荧光素酶报告基因。siRNA下调必需的细胞辅助因子有望在该系统中阻断HCV复制和荧光素酶活性。初步研究表明，高通量siRNA转染HCV复制子细胞系是可行的。我们讨论的方法命中选择，以及二次筛选步骤设计，以尽量减少假阳性结果的数量。丙型肝炎病毒感染可导致严重的肝脏疾病，包括肝硬化和肝癌。目前最好的丙型肝炎病毒治疗方法只对一半接受治疗的人有效，而且还可能导致严重的副作用。该项目希望发现病毒在细胞内繁殖所需的人类蛋白质，因为这些蛋白质可能成为新的HCV治疗方法的可能目标。

DESCRIPTION (provided by applicant): Project Summary: Chronic hepatitis C virus (HCV) infection affects about 3 percent of the world's population and HCV cirrhosis is now the leading indication for liver transplantation in the United States. The best current therapy for chronic HCV infection (combination peginterferon and ribavirin) leads to sustained virologic responses in only about half of all treated individuals. However, there are subgroups that have much lower rates of treatment response, such as African Americans and people with HIV coinfection. There is therefore a great need for novel HCV therapies. Small molecule inhibitors of HCV proteins are promising, but it is clear that viral resistance develops quickly to such agents. We propose to identify host genes that serve as viral replication cofactors, with the rationale that cellular cofactors may be targets for HCV therapy. For example, cyclophilin B is a host protein that has been shown to be involved in HCV replication, and small molecule inhibitors of cyclophilin B are potent inhibitors of HCV replication both in vitro and in vivo. In this application, we will screen a whole human genome siRNA library against a HCV replicon cell line that encodes a luciferase reporter gene. siRNA downregulation of essential cellular cofactors is expected to block HCV replication and luciferase activity in this system. Preliminary studies show that high-throughput siRNA transfection of a HCV replicon cell line is feasible. We discuss methods of hit selection, as well as secondary screening steps designed to minimize the number of false positive results. Public Health Relevance Hepatitis C virus infection can cause serious liver disease, including cirrhosis and liver cancer. The best current therapy for HCV only works in about half of all treated people, and also can cause serious side effects. This project hopes to discover the human proteins that the virus needs to reproduce inside cells, as these might turn out to be possible targets for new HCV therapies.