Hepatitis C Virus Trafficking in Infected Hepatocytes

描述（由申请人提供）：最近，丙型肝炎病毒（HCV）毒株在人肝癌细胞中具有传染性。这为研究病毒如何进入和离开肝细胞提供了第一个机会。我们提出了一种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病毒在感染细胞中的运输。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。我们组装了一个siRNA文库，其目标是参与所有已知膜运输途径的144个基因。我们对这个文库进行了研究，确定了48个HCV感染所需的宿主基因。这些基因聚集在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、高尔基结构和分泌途径的组成部分的特定途径中。我们利用分离病毒生命周期特定阶段的HCV细胞培养系统，确定了这些基因在病毒感染中的作用，包括HCV伪颗粒（HCV进入）和亚基因组HCV复制子（HCV复制）。然后，通过结合特异性抑制sirna与显微镜和生化方法来分析HCV在感染细胞中的运输，以确定HCV进入和输出的事件顺序。本文描述了遗传和生化方法来定义内吞运输和磷脂酰肌醇（PI）信号在HCV复制复合物形成中的功能。具体目标是：1。HCV进入细胞的细胞膜运输途径的分子特征。我们将定义HCV进入不同阶段的3种途径中对17个宿主基因的需求：确定hcv诱导膜重组建立复制的机制。我们将研究内噬运输和PI-4激酶IIIa在导致HCV复制复合体建立的膜重排中的作用。3. 确定对HCV颗粒成熟和输出重要的膜运输途径。我们将针对宿主基因参与出口表征病毒粒子出口和成熟的阶段。这些研究将阐明目前尚不清楚的病毒生命周期的主要阶段。此外，这些数据将进一步发展我们对使用高度相关和复杂探针（HCV）的人膜运输途径的组成和调控的理解。

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.