Structure and function of the HCV RNA replicase

HCV RNA 复制酶的结构和功能

• 批准号: 8026869
• 负责人: Charles M Rice
• 金额: $ 37.62万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8026869
• 关键词: Address; Animal Model; Antiviral Agents; Applications Grants; Binding; Biochemical; Biochemical Genetics; Biochemistry; Biological Assay; Cell Culture Techniques; Cell-Free System; Cells; Chimera organism; Chronic; Chronic Hepatitis C; Cirrhosis; Clinical; Complement; Complex; Coupled; Cultured Cells; Data; Development; Drug Delivery Systems; Elements; Enzymes; Genetic; Genome; Genomics; Goals; Health; Hepatitis C virus; Holoenzymes; Human; In Vitro; Individual; Infection; Integration Host Factors; Length; Life Cycle Stages; Link; Liver Failure; Liver diseases; Malignant Neoplasms; Mediating; Molecular; Molecular Biology; Molecular Cloning; Nonstructural Protein; Pathogenesis; Peptide Hydrolases; Polymerase; Polyproteins; Primary carcinoma of the liver cells; Process; Protease Domain; Proteins; RNA; RNA Binding; RNA chemical synthesis; RNA replication; RNA-Directed RNA Polymerase; Reagent; Replicon; Research; Resistance; Resolution; Risk; Role; Scheme; Structure; Surface; System; Therapeutic Intervention; Translations; Viral; Viral Proteins; Viral hepatitis; Virus; Virus Diseases; Virus Replication; X-Ray Crystallography; effective therapy; genetic analysis; genome-wide analysis; helicase; inhibitor/antagonist; insight; liver transplantation; novel; pathogen; protein complex; protein protein interaction; replicase; tool; viral RNA

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.

描述（由申请人提供）：丙型肝炎病毒（HCV）是肠胃外传播的非甲、非乙型病毒性肝炎的病原体。慢性感染使个体面临肝硬化、肝细胞癌和肝衰竭的风险，使慢性丙型肝炎成为肝移植的主要适应症。虽然 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希望，但耐药性的迅速出现表明需要额外的病毒靶标和抗病毒药物组合才能有效控制。我们建议研究复制酶复合物（HCV 复制的中心全酶）的结构和功能。进一步了解这种复合物的成分如何组合在一起，以及阐明每种成分的功能和机制，将有助于新型抗病毒药物的开发。我们将使用遗传分析与生化研究相结合来研究复制复合物内的蛋白质-蛋白质相互作用。这些研究将为建立对复制酶的结构理解提供一个背景。然后，我们的目标是阐明两种重要但神秘的病毒蛋白 NS3 解旋酶和 NS5A 的作用。这些蛋白质都是潜在极其有价值的药物靶点，但在 RNA 复制中的作用尚不清楚。我们将使用 X 射线晶体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 的原子分辨率结构，并研究其与 RNA 底物的复合物结构。我们还将尝试开发 HCV 的无细胞复制检测方法；目前缺乏此类系统是研究这种病毒的主要障碍。无细胞检测的出现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 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。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。