REPLICATION OF HEPATITIS C VIRUS RNA

丙型肝炎病毒 RNA 的复制

• 批准号: 6170633
• 负责人: Michael M.C. Lai
• 金额: $ 24.58万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6170633
• 关键词: DNA directed RNA polymerase; RNA biosynthesis; enzyme activity; genetic regulatory element; hepatitis C virus; laboratory rabbit; laboratory rat; nucleic acid sequence; protein protein interaction; tissue /cell culture; virus RNA; virus protein; virus replication

The overall objective of this application is to study the mechanism of hepatitis C virus (HCV) RNA replication and to establish a cell culture system for HCV RNA replication. Currently, the lack of an efficient replication system in tissue culture is one of the major obstacles in studying HCV. Our laboratory has expressed a recombinant HCV RNA polymerase, which is capable of replicating HCV RNA faithfully and appears independent of primers. Thus, this polymerase has the attributes expected of an authentic viral polymerase. This polymerase provides the opportunity to study HCV RNA replication in vitro and in vivo. Furthermore, it may allow the establishment of an HCV replication system in tissue culture. The following projects will be pursued: 1) Characterization of the HCV RNA polymerase activity in vitro. We will study the cis-acting sequences required for both (-)- and (+)-strand RNA synthesis in vitro, and the effects of the variable sequence at the 3'-UTR on RNA synthesis. We will also characterize the RNA products and the possible cellular factors on RNA synthesis in vitro. 2) Expression of a functional RNA polymerase in mammalian cells and establishment of an RNA replication system in cell culture. We will characterize the properties of the HCV polymerase expressed in mammalian cells. Once this polymerase is found to be active, we will use it as the basis for establishing an RNA replication system in cell culture, using an HCV defective-interfering (DI) RNA construct and full-length HCV RNA. 3) Characterization of the components of HCV RNA polymerase complex in the cells. We will study the interactions of other viral proteins, including ns4b and ns5a, with HCV polymerase. We will also characterize cellular proteins interacting with the polymerase. Finally we will study the effects of ns4b and ns5a on RNA synthesis in vivo.

本应用的总体目标是研究丙型肝炎病毒RNA复制的机制，并建立丙型肝炎病毒RNA复制的细胞培养体系。目前，组织培养中缺乏有效的复制系统是研究丙型肝炎病毒的主要障碍之一。我们实验室已经表达了一种能够忠实复制丙型肝炎病毒RNA的重组丙型肝炎病毒RNA聚合酶，并且不依赖于引物。因此，这种聚合酶具有真正的病毒聚合酶的预期属性。这种聚合酶为研究体外和体内的丙型肝炎病毒RNA复制提供了机会。此外，它还可以在组织培养中建立丙型肝炎病毒复制系统。将开展以下工作：1)体外鉴定丙型肝炎病毒RNA聚合酶活性。我们将研究在体外合成(-)和(+)链RNA所需的顺式作用序列，以及3‘-UTR处的可变序列对RNA合成的影响。我们还将表征RNA产物以及可能的细胞因素在体外合成RNA的作用。2)功能RNA聚合酶在哺乳动物细胞中的表达和细胞培养中RNA复制系统的建立。我们将描述在哺乳动物细胞中表达的丙型肝炎病毒聚合酶的特性。一旦发现这种聚合酶是活性的，我们将使用它作为在细胞培养中建立RNA复制系统的基础，使用丙型肝炎病毒缺陷干扰(DI)RNA结构和全长丙型肝炎病毒RNA。3)细胞内丙型肝炎病毒RNA聚合酶复合体的组成特征。我们将研究包括NS4B和NS5A在内的其他病毒蛋白与丙型肝炎病毒聚合酶的相互作用。我们还将描述与聚合酶相互作用的细胞蛋白质。最后，我们将研究NS4B和NS5A对体内RNA合成的影响。