Subversion of hepatocyte phosphoinositide metabolism by hepatitis C virus

描述（由申请人提供）：慢性丙型肝炎病毒（HCV）感染发生在所有暴露个体的70-80%中，影响世界人口的3%。虽然直接靶向病毒NS3-4A丝氨酸蛋白酶的新药今年已被批准临床使用，但仍需要更有效和耐受性更好的HCV治疗方法。我们的长期目标是确定宿主-HCV分子相互作用，并在此过程中确定新的HCV治疗药物。与迄今为止研究的所有其他正义单链RNA病毒一样，丙型肝炎病毒在感染细胞内诱导形成特定的膜结构（称为膜网）以进行自身复制。多个RNAi筛选已经确定了宿主蛋白磷脂酰肌醇4-激酶（PI 4-激酶）PI4KA在HCV复制中的关键作用，最有可能在膜网形成水平上。有趣的是，另外三种正义单链RNA病毒会破坏相关的PI - 4激酶PI4KB以进行自身复制。本研究的目的是了解宿主肝细胞蛋白PI4KA及其产物PI4 -磷酸（PI(4)P）如何支持HCV复制。我们的中心假设是HCV复制需要局部生成PI(4)P，而PI(4)P反过来又是将效应蛋白募集到新生膜网所必需的。该项目的具体目标是：(1)定义PI4KA的功能域及其调控；(2)确定HCV生命周期中磷酸肌醇依赖的步骤；(3)鉴定并表征HCV生命周期中PI4KA和PI(4)P的下游效应物。我们将使用各种生化、细胞和成像技术来完成拟议的研究。本文的创新之处在于：(1)本文提出的网络装配模型是新颖的；(2) PI4KA在宿主细胞和HCV复制中受到调控的机制尚未得到研究（目的1）；(3) PI(4)P和其他磷酸肌苷支持HCV复制的机制以前没有被研究过（目标2和3）；(4)磷酸肌苷的诱导和选择性耗竭（Aim 2）的使用以前并未用于研究病毒复制。我们希望这项研究能对公众健康有直接的好处，因为更好地了解PI4KA和宿主磷酸肌苷支持病毒复制的机制可能会带来治疗慢性HCV感染的新方法。更好地了解hcv -宿主关系是NIH肝病研究行动计划（目标B2a）的既定目标。此外，完成所提出的目标也将导致对PI4KA的生理功能和调控的新见解，这些领域目前知之甚少。

DESCRIPTION (provided by applicant): Chronic hepatitis C virus (HCV) infection develops in 70-80% of all exposed individuals and affects 3% of the world's population. While new drugs that directly target the viral NS3-4A serine protease have been approved for clinical use this year, there remains a need for more effective and better-tolerated HCV therapies. Our long- term goals are to define host-HCV molecular interactions and in so doing, to identify novel therapeutic agents for HCV therapy. HCV, like all other positive-sense single-stranded RNA viruses studied to date, induces the formation of specific membranous structures (called membranous webs) within infected cells for its own replication. Multiple RNAi screens have identified a critical role for a host protein phosphatidylinositol 4-kinase (PI 4-kinase) named PI4KA in HCV replication, most likely at the level of membranous web formation. Intriguingly, three other positive-sense single-stranded RNA viruses subvert the related PI 4-kinase PI4KB for their own replication. The objective of this proposal is to understand how the host hepatocyte protein PI4KA and its product PI 4-phosphate (PI(4)P) support HCV replication. Our central hypothesis is that HCV replication requires the local generation of PI(4)P, which in turn is necessary for the recruitment of effector proteins to the nascent membranous web. The specific aims of the project are to (1) Define the functional domains of PI4KA and its regulation; (2) Identify the phosphoinositide-dependent steps in the HCV life cycle; (3) Identify and characterize downstream effectors of PI4KA and PI(4)P in the HCV life cycle. We will use a variety of biochemical, cell-based, and imaging techniques to accomplish the proposed research. The proposed research is innovative as (1) the model of web assembly presented in this proposal is novel; (2) the mechanisms by which PI4KA is regulated in the host cell and in the context of HCV replication have not been studied (Aim 1); (3) The mechanisms by which PI(4)P and other phosphoinositides support HCV replication has not been previously pursued (Aims 2 and 3); (4) The use of inducible and selective depletion of phosphoinositides (Aim 2) has not been previously used to study viral replication. We expect that the proposed research may have direct public health benefits, as a better understanding of the mechanisms by which PI4KA and host phosphoinositides support viral replication may lead to novel approaches to the treatment of chronic HCV infection. A better understanding of the HCV-host relationship is a stated goal of the NIH Action Plan for Liver Disease Research (Goal B2a). Moreover, completion of the proposed aims will also lead to new insights into the physiological functions and regulation of PI4KA, areas that are currently poorly understood.