REPLICATION OF HEPATITIS C VIRUS RNA

本申请的总体目标是研究丙型肝炎病毒（HCV）RNA复制的机制，并建立用于HCV RNA复制的细胞培养系统。目前，缺乏有效的组织培养复制系统是研究HCV的主要障碍之一。 我们实验室表达了一种重组HCV RNA聚合酶，它能够忠实地复制HCV RNA，并且似乎不依赖于引物。 因此，该聚合酶具有真实病毒聚合酶的预期属性。 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，它可以允许在组织培养中建立HCV复制系统。 本研究将开展以下工作：1）HCV RNA聚合酶活性的体外研究。 我们将研究体外合成（-）-和（+）-链RNA所需的顺式作用序列，以及3 '-UTR可变序列对RNA合成的影响。 我们还将表征RNA产物和体外RNA合成的可能细胞因子。 2)哺乳动物细胞中功能性RNA聚合酶的表达和细胞培养中RNA复制系统的建立。 我们将描述哺乳动物细胞中表达的HCV聚合酶的特性。 一旦这种聚合酶被发现是活跃的，我们将使用它作为基础，在细胞培养中建立一个RNA复制系统，使用HCV缺陷干扰（DI）RNA构建体和全长HCV RNA。 3)细胞中HCV RNA聚合酶复合物组分的表征。 我们将研究其他病毒蛋白，包括ns 4 b和ns 5a，与HCV聚合酶的相互作用。 我们还将表征与聚合酶相互作用的细胞蛋白质。 最后，我们将研究ns 4 b和ns 5a在体内对RNA合成的影响。

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.