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R 01 CA 57973 -16“HCV RNA复制酶的结构和功能”的范围，该研究目前资助我们对丙型肝炎病毒（HCV）复制的研究。HCV感染全球超过1.3亿人，通常具有破坏性后果，包括肝硬化和肝细胞癌。我们目前正在研究构成HCV RNA复制复合物的单个病毒蛋白的结构和功能，我们还试图对有助于复制酶结构的病毒蛋白之间的相互作用进行编目，并最终在体外重建复制。在R 01 CA 57973 -16资金更新后的一个月内，我们在实现母基金目标方面取得了很大进展，包括准备描述NS 3解旋酶活性机制的手稿（Gu M和Rice CM，正在准备中）。我们现在建议扩大母基金的目标，包括识别和表征功能性宿主因子与HCV复制酶的相互作用。这项新的研究将补充我们目前资助的病毒蛋白质之间的遗传、生物化学和结构相互作用的研究，并加速对催化HCV复制的中央多蛋白复合物的更完整理解。我们将通过对感染细胞内天然复合物中的单个病毒蛋白进行下拉测定，来严重地将我们的研究偏向于鉴定功能性相互作用。为了做到这一点，我们将发现每个HCV基因内插入标签的允许位点，我们将优化条件以下拉病毒蛋白和相关因子，我们将独立验证并对每个相互作用进行机制研究。所申请的资金将用于启动该项目，方法是购买重要的试剂组，并支持一名目前未获得资助的博士后助理，部分资助一名博士。学生，并雇用一名额外的技术人员。因此，拟议的支出将进一步实现《恢复法》的目标，同时大大增加我们对HCV复制酶复合体的了解。 公共卫生相关性：丙型肝炎病毒（HCV）是一种嗜肝性人类病毒，目前感染全球约1.3亿人。我们正在研究HCV复制其RNA基因组并产生新的感染性颗粒的机制，希望确定可用于开发抗病毒药物的相互作用。目前资助的研究正在调查病毒蛋白质之间的相互作用，这是重要的复制复杂的功能;这里提出的新研究扩展了我们的研究，探索细胞因子在HCV传播的重要性。