REPLICATION OF HEPATITIS C VIRUS RNA

丙型肝炎病毒 RNA 的复制

• 批准号: 6534244
• 负责人: Michael M.C. Lai
• 金额: $ 23.1万
• 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 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6534244
• 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.

本应用程序的总体目的是研究丙型肝炎病毒（HCV） RNA复制的机制，并建立用于HCV RNA复制的细胞培养体系。目前，缺乏高效的组织培养复制系统是HCV研究的主要障碍之一。我们实验室已经表达了一种重组HCV RNA聚合酶，该酶能够忠实地复制HCV RNA，并且不依赖于引物。因此，这种聚合酶具有预期的真实病毒聚合酶的属性。这种聚合酶提供了在体外和体内研究HCV RNA复制的机会。此外，它可能允许在组织培养中建立HCV复制系统。研究方向：1)体外HCV RNA聚合酶活性的表征。我们将研究体外合成（-）链和（+）链RNA所需的顺式作用序列，以及3'- utr上的可变序列对RNA合成的影响。我们还将表征RNA产物和可能的细胞因子在体外RNA合成。2)在哺乳动物细胞中表达一种功能性RNA聚合酶，并在细胞培养中建立RNA复制系统。我们将描述在哺乳动物细胞中表达的HCV聚合酶的特性。一旦发现这种聚合酶具有活性，我们将利用它作为在细胞培养中建立RNA复制系统的基础，使用HCV缺陷干扰（DI） RNA构建物和全长HCV RNA。3)细胞中HCV RNA聚合酶复合物组分的鉴定。我们将研究其他病毒蛋白（包括ns4b和ns5a）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。最后，我们将研究ns4b和ns5a对体内RNA合成的影响。