Structure and function of the HCV RNA replicase

描述（由申请人提供）：丙型肝炎病毒（HCV）是肠道外传播的非甲、非乙型病毒性肝炎的病原体。慢性感染使个体处于肝硬化、肝细胞癌和肝功能衰竭的发展风险中，使慢性丙型肝炎成为肝移植的主要指征。虽然HCV特异性蛋白酶和聚合酶抑制剂在早期临床开发中显示出前景，但快速出现的耐药性表明需要额外的病毒靶点和抗病毒药物的组合来进行有效控制。我们建议调查的复制酶复合物，丙型肝炎病毒复制的中央全酶的结构和功能。增加对这种复合物的组分如何结合在一起的理解，以及澄清每个组分的功能和机制，将促进新型抗病毒药物的开发。我们将使用遗传分析与生物化学研究相结合的方法来研究复制复合物中的蛋白质-蛋白质相互作用。这些研究将提供一个背景下，建立一个结构的理解复制酶。然后，我们的目标是阐明两个重要的，但神秘的病毒蛋白，NS 3解旋酶和NS 5A的作用。这些蛋白质都是潜在的极有价值的药物靶点，在RNA复制中具有未知的作用。我们将使用X射线晶体学和互补遗传学方法研究NS 3解旋酶活性的机制以及这种酶在病毒生命周期中的作用。我们将追求全长NS 5A的原子分辨率结构，并研究其与RNA底物复合的结构。我们还将尝试开发无细胞复制检测HCV;目前缺乏这样的系统是研究这种病毒的主要障碍。无细胞分析的可用性将使我们能够将结构，生物化学和遗传数据与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HCV RNA复制的机制，并发现治疗干预的新途径。公共卫生相关性：丙型肝炎病毒是导致包括癌症在内的肝脏疾病的主要原因。该提案旨在研究复制病毒基因组的蛋白质复合物的结构和功能。研究结果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.