Hepatitis C Virus Trafficking in Infected Hepatocytes

描述（由申请人提供）：最近，鉴定了在人肝癌细胞中具有感染性的丙型肝炎病毒（HCV）毒株。这为研究病毒如何进入和离开肝细胞提供了第一次机会。我们提出了一个组合分析来剖析细胞膜运输途径在HCV感染中的作用。我们将整合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感染细胞中的病毒运输。这将是特别富有成效的，因为HCV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。组装靶向参与所有已知膜运输途径的144个基因的siRNA文库。我们已经查询了这个文库，并确定了HCV感染所需的48个宿主基因。这些基因聚集成网格蛋白介导的内吞作用、细胞骨架重组、内吞运输、磷脂酰肌醇（PI）信号传导、膜重组、高尔基体结构和分泌途径组分的特定途径。我们已经分配了这些基因在病毒感染中的作用，使用HCV细胞培养系统分离病毒生命周期的特定阶段，包括HCV假颗粒（HCV进入）和亚基因组HCV复制子（HCV复制）。然后将通过将特异性抑制性siRNA与显微镜和生物化学方法相结合来分析感染细胞中的HCV运输，以确定HCV进入和排出的事件顺序。遗传和生物化学的方法来定义的功能内吞运输和磷脂酰肌醇（PI）信号在HCV复制复合物的形成。具体目标是：1. HCV进入细胞的膜运输途径的分子表征。我们将定义HCV进入不同阶段的3个途径中17个宿主基因的要求：2。确定HCV诱导的膜重组建立复制的机制。我们将研究内吞运输和PI-4激酶IIIa在导致HCV复制复合物建立的膜重排中的功能。3.识别对HCV颗粒成熟和排出重要的膜运输途径。我们将靶向宿主基因参与出口，以表征病毒体出口和成熟的阶段。 这些研究将阐明病毒生命周期的主要阶段，目前还不清楚。此外，这些数据将进一步发展我们的理解到人类膜运输途径的组件和调节使用高度相关和复杂的探针（HCV）。 公共卫生相关性：1.7亿人感染丙型肝炎病毒（HCV），可导致肝癌和终末期肝病。该提案调查了人类基因在丙型肝炎病毒从受感染的肝细胞进入、复制和退出中的作用。这些研究将调查HCV复制的机制，并确定可能影响HCV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.