RNA-dependent RNA Synthesis by the Hepatitis C Virus

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)感染约2%的世界人口，是美国肝脏移植的主要原因。根据从艾滋病等疾病中吸取的经验教训，丙型肝炎病毒RNA复制是抗病毒开发的一个有前途的目标。然而，对所有具有正链RNA基因组的病毒的复制知之甚少，特别是在生化水平上。Kao实验室研究的总体目标是了解RNA病毒的复制机制。 这个项目的目标是建立关于丙型肝炎病毒复制复合体亚单位的知识，重点是蛋白质-RNA和蛋白质-蛋白质相互作用。这是Kao实验室过去六年研究的延伸，在Kao实验室，使用生化、生物物理和基于细胞的方法检测了丙型肝炎病毒聚合酶和丙型肝炎病毒蛋白解旋酶的一些基本性质。这项研究可以分为几个相关的分目标，这些目标将： 1.鉴定和验证丙型肝炎病毒RdRp中与底物NTPs、模板RNA相互作用的残基以及在丙型肝炎病毒RNA合成的不同阶段的生物学重要性。 2.阐明丙型肝炎病毒RdRp与新生RNA之间的相互作用，鉴定和表征新生RNA输出通道。 3.检测蛋白质-蛋白质和蛋白质-RNA与丙型肝炎病毒复制酶其他复制酶相关亚单位的相互作用，并检测这些相互作用对细胞中丙型肝炎病毒复制酶形成的影响。 4.利用电子显微镜获得蛋白质复合体的图像并重建其结构。 这一建议的结果将促进对所有正链RNA病毒依赖病毒RNA合成RNA的机制的理解。这些知识还可以用来比较所有依赖于模板的(病毒和细胞)聚合酶的作用机制。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) infects approximately 2% of the world's population and is the primary cause of liver transplants in the United States. Based on lessons learned from diseases such as AIDS, HCV RNA replication is a promising target for antiviral development. However, the replication of all viruses with plus-strand RNA genomes is poorly understood, especially at the biochemical level. The overall goal of the research in the Kao lab is to understand mechanism of RNA virus replication. The goal for this project is to build knowledge about the subunits of the HCV replication complex, with emphasis on protein-RNA, and protein-protein interactions. This is an extension of the past six years of research in the Kao lab, where a number of basic properties of the HCV polymerase and the HCV protease-helicase have been examined using biochemical, biophysical, and cell-based methods. The research can be partitioned into several related subaims that will: 1. Identify and validate the biological importance of the residues in the HCV RdRp that interacts with the substrate NTPs, the template RNA, and during different stages of HCV RNA synthesis. 2. Elucidate the interactions between the HCV RdRp and the nascent RNA and identify and characterize the nascent RNA exit channel. 3. Examine the protein-protein and protein-RNA interactions with other replicase- associated subunits of the HCV replicase and examine effects of the interactions on HCV replicase formation in cells. 4. Obtain images of the protein complexes using electron microscopy and reconstruct their structures. Results from this proposal will advance the understanding of the mechanism of viral RNA-dependent RNA synthesis for ALL positive-strand RNA viruses. The knowledge can also be used to compare the mechanisms of action of all template-dependent (both viral and cellular) polymerases.