Structure and function of the HCV RNA replicase

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是由肠道外传播的非甲非乙型病毒性肝炎的病原体。慢性感染使个人面临发展为肝硬变、肝细胞癌和肝功能衰竭的风险，使慢性丙型肝炎成为肝移植的主要适应症。虽然丙型肝炎病毒特异的蛋白酶和聚合酶抑制剂在早期临床开发中显示出希望，但耐药性的迅速出现表明，将需要更多的病毒靶点和抗病毒药物的组合来有效控制。我们建议研究复制酶复合体的结构和功能，复制酶复合体是丙型肝炎病毒复制的中心全酶。进一步了解这种复合体的成分是如何结合在一起的，以及澄清每个成分的功能和机制，将有助于新型抗病毒药物的开发。我们将使用遗传分析和生化研究相结合的方法来研究复制复合体中的蛋白质-蛋白质相互作用。这些研究将提供一个背景，在其中建立对复制酶的结构理解。然后，我们的目标是阐明两个重要但神秘的病毒蛋白，NS3解旋酶和NS5A的作用。这两种蛋白质都是潜在的极有价值的药物靶点，在RNA复制中的作用尚不清楚。我们将利用X射线结晶学和互补遗传方法研究NS3解旋酶活性的机制以及该酶在病毒生命周期中的作用。我们将追求全长NS5A的原子分辨结构，并在与RNA底物的络合物中研究其结构。我们还将尝试开发丙型肝炎病毒的无细胞复制分析方法；目前缺乏这样的系统是研究这种病毒的主要障碍。无细胞测试的可用性将使我们能够将结构、生化和遗传数据与RNA合成的特定步骤联系起来。通过这些研究，我们希望开始了解丙型肝炎病毒RNA复制的机制，并发现治疗干预的新途径。公共卫生相关性：丙型肝炎病毒是包括癌症在内的肝病的主要原因。这项提议旨在研究复制病毒基因组的蛋白质复合体的结构和功能。由此产生的发现应该有助于开发更有效的治疗方法，旨在根除这种致命的病毒性疾病。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is the etiologic agent of parenterally transmitted non-A, non-B viral hepatitis. Chronic infection puts individuals at risk for the development of cirrhosis, hepatocellular carcinoma, and liver failure, making chronic hepatitis C the leading indication for liver transplantation. While HCV-specific protease and polymerase inhibitors are showing promise in early clinical development, rapid emergence of resistance indicates that additional viral targets and combinations of antivirals will be needed for effective control. We propose to investigate the structure and function of the replicase complex, the central holoenzyme of HCV replication. Increased understanding of how the components of this complex come together, as well as clarifying the functions and mechanisms of each constituent, will facilitate the development of novel antiviral drugs. We will use genetic analyses complemented with biochemical studies to investigate protein-protein interactions within the replication complex. These studies will provide a context in which to build a structural understanding of the replicase. We then aim to elucidate the roles of two important but enigmatic viral proteins, the NS3 helicase and NS5A. These proteins, both potentially extremely valuable drug targets, have as-yet-unknown roles in RNA replication. We will investigate the mechanism of NS3 helicase activity and the roles of this enzyme in the viral life cycle using X-ray crystallography and complementary genetic approaches. We will pursue an atomic-resolution structure of full-length NS5A, as well as investigate its structure in complex with an RNA substrate. We will also attempt to develop cell-free replication assays for HCV; the current lack of such systems is a major roadblock to studies of this virus. Availability of a cell-free assay would allow us to relate structural, biochemical, and genetic data to specific steps of RNA synthesis. Through these studies, we hope to begin to understand mechanisms of HCV RNA replication and to uncover novel avenues for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Hepatitis C virus is a leading cause of liver disease including cancer. This proposal aims to study the structure and function of the protein complex that replicates the virus genome. The resulting findings should aid development of more effective treatments aimed at eradicating this deadly viral disease.