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

描述(申请人提供)：项目摘要：慢性丙型肝炎病毒(丙型肝炎病毒)感染影响大约3%的世界人口，丙型肝炎病毒肝硬变目前是美国肝移植的主要适应症。目前治疗慢性丙型肝炎病毒感染的最佳疗法(联合使用聚乙二醇干扰素和利巴韦林)在所有接受治疗的患者中只有大约一半的人有持续的病毒学应答。然而，也有一些亚群的治疗应答率要低得多，例如非裔美国人和艾滋病毒合并感染者。因此，非常需要新的丙型肝炎病毒治疗方法。丙型肝炎病毒蛋白的小分子抑制剂很有希望，但很明显，病毒对这种药物的耐药性发展很快。我们建议识别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丙型肝炎病毒治疗的靶点。例如，亲环素B是一种宿主蛋白，已被证明参与了丙型肝炎病毒的复制，而亲环素B的小分子抑制剂在体外和体内都是有效的丙型肝炎病毒复制抑制剂。在这项应用中，我们将针对编码荧光素酶报告基因的丙型肝炎病毒复制子细胞系筛选完整的人类基因组siRNA文库。SiRNA下调必要的细胞辅因子有望阻止该系统中的丙型肝炎病毒复制和荧光素酶活性。初步研究表明，高通量siRNA转导丙型肝炎病毒复制子细胞系是可行的。我们讨论了命中选择的方法，以及旨在将假阳性结果的数量降至最低的二次筛选步骤。与公共卫生相关丙型肝炎病毒感染可导致严重的肝病，包括肝硬变和肝癌。目前最好的治疗丙型肝炎病毒的方法只对大约一半的接受治疗的人有效，而且还会引起严重的副作用。该项目希望发现病毒在细胞内复制所需的人类蛋白，因为这些蛋白可能成为新的丙型肝炎疗法的靶点。

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.