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NS 3 -4A丝氨酸蛋白酶的新药今年已被批准用于临床，但仍然需要更有效和更耐受的HCV治疗。我们的长期目标是确定宿主-HCV分子相互作用，并在此过程中鉴定用于HCV治疗的新型治疗剂。丙型肝炎病毒，像所有其他的正义单链RNA病毒研究的日期，诱导形成特定的膜结构（称为膜网）内感染的细胞，为自己的复制。多个RNAi筛选已经确定了一种名为PI 4KA的宿主蛋白磷脂酰肌醇4-激酶（PI 4-kinase）在HCV复制中的关键作用，最有可能是在膜网形成水平。有趣的是，其他三种正义单链RNA病毒破坏了相关的PI 4-激酶PI 4KB以进行自身复制。本提案的目的是了解宿主肝细胞蛋白PI 4KA及其产物PI 4-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PI（4）P的局部生成，而PI（4）P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PI 4KA及其调控的功能域;（2）识别HCV生命周期中的磷酸肌醇依赖性步骤;（3）识别和表征HCV生命周期中PI 4KA和PI（4）P的下游效应物。我们将使用各种生物化学，细胞为基础的，和成像技术来完成拟议的研究。本研究具有创新性，因为（1）本研究中提出的网络组装模型是新颖的;（2）尚未研究PI 4 KA在宿主细胞中和HCV复制背景下的调节机制（目的1）;（3）PI（4）P和其他磷酸肌醇支持HCV复制的机制先前尚未研究（目的2和3）;（4）使用可诱导的和选择性的磷酸肌醇耗竭（目的2）以前没有用于研究病毒复制。我们预计，拟议的研究可能会有直接的公共卫生利益，作为一个更好地了解PI 4KA和宿主磷酸肌醇支持病毒复制的机制可能会导致新的方法来治疗慢性HCV感染。更好地了解HCV-宿主关系是NIH肝病研究行动计划的既定目标（目标B2 a）。此外，完成所提出的目标也将导致对PI 4KA的生理功能和调节的新见解，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.