Structure and function of the HCV RNA replicase

描述（由申请人提供）：我们建议扩大R01 CA57973-16“HCV RNA复制酶的结构和功能”的范围，该项目目前资助我们对丙型肝炎病毒（HCV）复制的研究。全世界有超过1.3亿人感染丙型肝炎病毒，其后果往往是毁灭性的，包括肝硬化和肝细胞癌。我们目前正在研究组成HCV RNA复制复合体的单个病毒蛋白的结构和功能，我们也在尝试编目有助于复制酶结构的病毒蛋白之间的相互作用，并最终在体外重建复制。在R01 CA57973-16基金续期的一个月里，我们在实现母基金目标方面取得了很好的进展，包括准备了一篇描述NS3解旋酶活性机制的手稿（Gu M和Rice CM，正在准备中）。我们现在提议扩大母体资助的目标，包括鉴定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之间的遗传、生化和结构相互作用的研究，并加速对催化丙型肝炎病毒复制的中心多蛋白复合体的更全面了解。我们将在很大程度上偏向于通过对感染细胞内天然复合物的单个病毒蛋白进行下拉分析来鉴定功能相互作用。为了做到这一点，我们将在每个HCV基因中发现允许标签插入的位点，我们将优化条件以拉下病毒蛋白和相关因子，我们将独立验证并对每种相互作用进行机制研究。申请的资金将用于启动该项目，通过购买重要的试剂集，支持一名目前没有资金的博士后，部分资助一名博士生，并雇用一名额外的技术人员。因此，拟议的支出将进一步推动《康复法案》的目标，同时大大增加我们对丙型肝炎病毒复制酶复合体的了解。

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.