Hepatitis C Virus Trafficking in Infected Hepatocytes

描述(申请人提供)：最近，丙型肝炎病毒(丙型肝炎病毒)株被鉴定为在人肝癌细胞中具有感染性。这为研究病毒如何进入和离开肝细胞提供了第一个机会。我们提出了一个组合分析来剖析细胞膜运输途径在丙型肝炎病毒感染中的作用。我们将结合RNA干扰(RNAi)分析、活细胞显微镜、病毒遗传学和生物化学来探测病毒在感染细胞中的传播。这将是特别有成效的，因为丙型肝炎病毒在其生命周期的每一步都在一系列复杂的相互作用中使用膜运输途径。组装了一个siRNA文库，该文库针对参与所有已知的膜转运途径的144个基因。我们对这个文库进行了查询，鉴定出了48个丙型肝炎病毒感染所需的宿主基因。这些基因聚集在特定的胞内吞噬、细胞骨架重组、胞内转运、磷脂酰肌醇(PI)信号转导、膜重组、高尔基体结构和分泌途径的组成部分。我们使用分离病毒生命周期特定阶段的丙型肝炎病毒细胞培养系统，为这些基因分配了在病毒感染中的作用，包括丙型肝炎病毒假颗粒(丙型肝炎病毒进入)和亚基因组丙型肝炎病毒复制子(丙型肝炎病毒复制)。然后，通过结合特定的抑制性siRNAs与显微镜和生化方法来分析丙型肝炎病毒在感染细胞中的运输，以确定丙型肝炎病毒进出的事件顺序。描述了遗传和生化方法来确定内吞转运和磷脂酰肌醇(PI)信号转导在形成丙型肝炎病毒复制复合体中的功能。具体目标是：1.丙型肝炎病毒进入细胞的膜转运途径的分子特征。我们将在3条途径中确定丙型肝炎病毒进入不同阶段对17个宿主基因的要求：2.确定丙型肝炎病毒诱导的膜重组建立复制的机制。我们将研究内吞转运和PI-4激酶IIIa在导致建立丙型肝炎病毒复制复合体的膜重排中的作用。3.确定对丙型肝炎病毒颗粒成熟和排出具有重要作用的膜转运途径。我们将针对参与病毒出口的宿主基因来描述病毒粒子出口和成熟的阶段。这些研究将阐明病毒生命周期的主要阶段，目前这些阶段的特征很差。此外，这些数据将进一步发展我们对使用高度相关和复杂的探针(丙型肝炎病毒)的人膜运输途径的组成和调控的理解。 公共卫生相关性：1.7亿人感染丙型肝炎病毒(丙型肝炎病毒)，可导致肝癌和终末期肝病。这项建议调查了人类基因在丙型肝炎病毒从受感染的肝细胞进入、复制和离开中的作用。这些研究将研究丙型肝炎病毒复制的机制，并确定可能影响丙型肝炎病毒感染人群如何发展肝病的人类基因的关键贡献。

DESCRIPTION (provided by applicant): Recently, hepatitis C virus (HCV) strains that are infectious in human hepatoma cells were identified. This provides the first opportunity to study how the virus enters and exits liver cells. We propose a combinatorial analysis to dissect the roles of cellular membrane trafficking pathways in HCV infection. We will integrate RNA interference (RNAi) analysis, live cell microscopy, viral genetics, and biochemistry to probe viral trafficking in the infected cell. This will be especially fruitful, since HCV uses membrane trafficking pathways at each step of its life cycle in a series of complex interactions. An siRNA library was assembled that targets 144 genes that participate in all known membrane trafficking pathways. We have interrogated this library and identified 48 host genes that are required for HCV infection. These genes cluster into specific pathways of clathrin-mediated endocytosis, cytoskeletal re- organization, endocytic trafficking, phosphatidyl inositol (PI) signaling, membrane re-organization, Golgi structure, and components of the secretory pathway. We have assigned roles for these genes in viral infection using HCV cell culture systems that isolate specific stages of the viral life cycle, including HCV pseudo-particles (HCV entry) and sub-genomic HCV replicons (HCV replication). HCV trafficking in the infected cell will then be analyzed by combining the specific inhibitory siRNAs with microscopy and biochemical approaches to determine the sequence of events in HCV entry and egress. Genetic and biochemical approaches are described to define the functions of endocytic trafficking and phosphatidyl inositol (PI) signaling in the formation of HCV replication complexes. The specific aims are: 1. Molecular characterization of membrane trafficking pathways for HCV entry into cells. We will define the requirements for 17 host genes in 3 pathways for the distinct stages of HCV entry: 2. Identify the mechanism of HCV-induced membrane re-organization for establishing replication. We will investigate the function of endocytic trafficking and PI-4 kinase IIIa in membrane re-arrangements leading to the establishment of HCV replication complexes. 3. Identify membrane trafficking pathways important for HCV particle maturation and egress. We will target the host genes involved in egress to characterize the stages of virion egress and maturation. These studies will illuminate major stages of the viral life cycle that are currently poorly characterized. Additionally, these data will further develop our understanding into the components and regulation of human membrane trafficking pathways using a highly relevant and complex probe (HCV). PUBLIC HEALTH RELEVANCE: 170 million people are infected with hepatitis C virus (HCV), which can lead to liver cancer and end stage liver disease. This proposal investigates the roles of human genes in the entry, replication, and exit of hepatitis C virus from infected liver cells. These studies will investigate the mechanism of HCV replication and identify key contributions of human genes that may affect how the HCV-infected population develops liver disease.