Structure and function of the HCV RNA replicase

HCV RNA 复制酶的结构和功能

• 批准号: 7811290
• 负责人: Charles M Rice
• 金额: $ 56.11万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7811290
• 关键词: Affinity; Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; Architecture; Biochemical; Biological Assay; Cataloging; Catalogs; Cells; Complement; Complex; Databases; Development; Doctor of Philosophy; Ensure; Epitopes; Expenditure; Follow-Up Studies; Funding; Genes; Genetic; Genome; Hepatitis C virus; Human Virus; In Vitro; Individual; Integration Host Factors; Investigation; Life Cycle Stages; Liver; Liver Cirrhosis; Manuscripts; Maps; Mass Spectrum Analysis; Mediating; Multiprotein Complexes; Pathway interactions; Pharmaceutical Preparations; Play; Precipitation; Preparation; Primary carcinoma of the liver cells; Process; Proteins; RNA; RNA replication; RNA-Directed RNA Polymerase; Reagent; Recovery; Research; Rice; Role; Site; Small Interfering RNA; Staging; Structure; Students; Technology; United States National Institutes of Health; Viral; Viral Genes; Viral Genome; Viral Nonstructural Proteins; Viral Proteins; Virion; Virus Replication; cohort; drug development; genome-wide; helicase; parent grant; particle; public health relevance; reconstitution; replicase; response; viral RNA

DESCRIPTION (provided by applicant): We propose to expand the scope of R01 CA57973-16 "Structure and function of the HCV RNA replicase", which currently funds our studies of hepatitis C virus (HCV) replication. HCV infects over 130 million people worldwide, with often devastating consequences including liver cirrhosis and hepatocellular carcinoma. We are currently investigating the structures and functions of individual viral proteins that make up the HCV RNA replication complex, we are also attempting to catalog interactions between viral proteins that contribute to the architecture of the replicase, and ultimately to reconstitute replication in vitro. In the one month since the renewal of R01 CA57973-16 funding, we have made excellent progress towards the aims of the parent grant, including preparing a manuscript describing the mechanism of NS3 helicase activity (Gu M and Rice CM, in preparation). We are now proposing to extend the aims of the parent grant to include the identification and characterization of functional host factor interactions with the HCV replicase. This new line of investigation will complement our currently funded studies of genetic, biochemical, and structural interactions between viral proteins, and accelerate progress towards a more complete understanding of the central multiprotein complex catalyzing HCV replication. We will heavily bias our studies towards the identification of functional interactions by performing pull-down assays of individual viral proteins from native complexes within an infected cell. To do this we will discover permissive sites for tag insertion within each HCV gene, we will optimize conditions to pull-down the viral protein and associated factors, and we will independently validate and perform mechanistic studies on each interaction. The requested funding will be used to jump-start this project by acquiring significant reagent sets and by supporting one currently unfunded postdoctoral associate, partially funding one Ph.D. student, and hiring an additional technician. The proposed expenditures will thereby further the aims of the Recovery Act while dramatically increasing our understanding of the HCV replicase complex. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a liver-tropic human virus that currently infects approximately 130 million people worldwide. We are studying the mechanisms by which HCV replicates its RNA genome and produces new infectious particles, with the hopes of identifying interactions that can be exploited for the development of anti-viral drugs. Currently funded studies are investigating the interactions between viral proteins that are important for replication complex function; the new studies proposed here expand our research to explore the importance of cellular factors in HCV propagation.

描述（由申请人提供）：我们建议扩大R01 CA57973-16的范围“ HCV RNA复制酶的结构和功能”，该酶目前为我们对丙型肝炎病毒（HCV）复制的研究提供了资金。 HCV在全球范围内感染了超过1.3亿人，经常造成灾难性的后果，包括肝硬化和肝细胞癌。我们目前正在研究构成HCV RNA复制复合复合物的个体病毒蛋白的结构和功能，我们还试图在病毒蛋白之间进行分类的病毒蛋白之间的相互作用，这些病毒蛋白有助于复制酶的结构，并最终在体外重新构成复制。自从R01 CA57973-16资金续签以来的一个月中，我们取得了良好的进步，朝着父母赠款的目标方面取得了长足的进步，包括准备一份手稿，描述了NS3解旋酶活性的机制（GU M和RICE CM，准备准备）。现在，我们建议扩展父授予的目标，以包括功能宿主因子与HCV复制酶相互作用的识别和表征。这项新的研究系列将补充我们目前对病毒蛋白之间遗传，生化和结构相互作用的资助研究，并加速进步，以更完整地理解中央多蛋白复合物催化HCV的复制。我们将通过从感染细胞内的天然复合物中对单个病毒蛋白进行下拉测定法，这将严重偏向于鉴定功能相互作用的鉴定。为此，我们将发现每个HCV基因内的TAG插入的允许位点，我们将优化条件以下拉病毒蛋白和相关因素，我们将独立验证和对每种相互作用进行机械研究。要求的资金将通过获取重要的试剂集并支持当前无资金的博士后助理，部分资助一位博士学位来开始该项目开始该项目。学生，并聘请另一位技术人员。拟议的支出将进一步进一步，同时大大提高了我们对HCV复制酶复合物的理解。 公共卫生相关性：丙型肝炎病毒（HCV）是一种肝疗法的人类病毒，目前感染了全球约1.3亿人。我们正在研究HCV复制其RNA基因组并产生新的传染性颗粒的机制，希望鉴定可以利用用于开发抗病毒药物的相互作用。目前，资助的研究正在研究病毒蛋白之间对复制复杂功能很重要的相互作用。这里提出的新研究扩大了我们的研究，以探讨细胞因素在HCV传播中的重要性。