HEPATITIS C VIRUS TRAFFICKING IN INFECTED HEPATOCYTES

最近，丙型肝炎病毒（HCV）株是感染人肝癌细胞被确定。这 首次提供了研究病毒如何进出肝细胞的机会。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， 生物化学来探测受感染细胞中的病毒运输。这将是特别富有成效的，因为HCV 在一系列复杂的相互作用中，在其生命周期的每一步都使用膜运输途径。一个 组装siRNA文库，其靶向参与所有已知膜运输的144个基因 途径。我们对该文库进行了分析，并鉴定了HCV所需的48个宿主基因 感染这些基因聚集成网格蛋白介导的内吞作用、细胞骨架重建和细胞凋亡的特定途径。 组织，内吞运输，磷脂酰肌醇（PI）信号，膜重组，高尔基体 结构和分泌途径的组成部分。我们已经确定了这些基因在病毒中的作用， 使用HCV细胞培养系统分离病毒生命周期的特定阶段的感染，包括HCV 假颗粒（HCV进入）和亚基因组HCV复制子（HCV复制）。丙型肝炎病毒的贩运 然后通过将特异性抑制性siRNA与显微镜结合来分析感染的细胞， 生物化学方法来确定HCV进入和排出的事件顺序。遗传和 描述了生物化学方法来定义内吞运输和磷脂酰的功能。 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在膜重排中的功能 导致HCV复制复合体的建立。 3.识别对HCV颗粒成熟和排出重要的膜运输途径。我们 将靶向参与排出的宿主基因，以表征病毒体排出和成熟的阶段。 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对药物成分和调节的理解。 人膜运输途径使用高度相关和复杂的探针（HCV）。

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).