Role of Lipids in Hepatitis C Virus Maturation

简介（由申请人提供）：约3%的世界人口感染丙型肝炎病毒（HCV）。丙型肝炎病毒感染通常导致慢性肝炎，约三分之一的患者发展为肝硬化，其中少数发展为肝细胞癌。这种感染还与脂肪肝有关。HCV在由病毒和宿主细胞因子组成的核糖核蛋白复合体（RNP）中复制其RNA基因组。一个这样的宿主因子，称为氧甾醇结合蛋白（OSBP），由我们鉴定亲和力为基础的方法。随后的研究表明OSBP是HCV分泌/释放所必需的，对细胞内RNA复制无明显影响。它属于一组蛋白质被称为磷脂酰肌酸-4磷酸（PI4P）相互作用蛋白。在本应用中，我们重点研究了OSBP和另外两个pi4p相互作用蛋白CERT和GOLPH3在HCV成熟/分泌通路中的作用。高尔基激酶PKD在HCV成熟中的作用也将被研究。HCV成熟/分泌，可能发生在高尔基腔室，被广泛认为与极低密度脂蛋白（VLDL）颗粒有关。我们建议在OSBP和VLDL分泌途径的背景下，通过共聚焦激光显微镜和免疫荧光来表征HCV通过高尔基室的成熟过程。VLDL颗粒通过专门的VLDL运输囊泡（vtv）分泌。利用已建立的分离vtv的生化分离程序，我们建议确定HCV成分与vtv的关系。表征含HCV病毒粒子成分的HCV感染细胞中的vtv对理解HCV形态发生具有重要意义。确定参与这种组装/分泌过程的宿主因子也将为设计新的抗病毒策略开辟新的途径。这些研究将揭示HCV成熟、分泌和出芽过程的独特机制，并为其他RNA病毒提供独特的模型。

DESCRIPTION (provided by applicant): About 3% of world population is infected with Hepatitis C virus (HCV). HCV infection often leads to chronic hepatitis and about one third of patients develop cirrhosis and a minority of those develops hepatocellular carcinoma. The infection is also associated with fatty liver disease. HCV replicates its RNA genome within ribonucleoprotein complexes (RNP) consisting of viral and host cellular factors. One such host factor, termed oxysterol binding protein (OSBP) was identified by an affinity based approach by us. Subsequent studies revealed that OSBP was necessary for HCV secretion/release with no obvious effect on intracellular RNA replication. It belongs to group of proteins referred to as phosphatidyinosito-4 phosphate (PI4P)-interacting proteins. In this application, we focus on the investigation of OSBP and two other PI4P-interacting proteins, CERT and GOLPH3 in HCV maturation/secretion pathway. Role of Golgi kinase PKD in HCV maturation will also investigated. HCV maturation/secretion, which likely occurs in the Golgi compartment, is widely believed to occur in association with very low density lipoprotein (VLDL) particles. We propose to characterize the HCV maturation process through Golgi compartments by confocal laser microscopy and immunofluorescence in the context of OSBP and VLDL secretory pathway. VLDL particles are secreted via specialized VLDL transport vesicles (VTVs). Using an established biochemical fractionation procedure of isolation of VTVs, we propose to determine the association of HCV components with VTVs. Characterization of VTVs in HCV infected cells containing HCV virion components is of fundamental importance in understanding the HCV morphogenesis. Identification of host factors involved in this assembly/secretion process will also open new avenues for designing novel antiviral strategies. These studies will reveal unique insights into the mechanisms of HCV maturation, secretion and budding processes and provide a unique model for other RNA viruses. PUBLIC HEALTH RELEVANCE: HCV infections progresses to cirrhosis, steatosis, and liver cancer and are the leading indications for liver transplants thus representing a major public health burden. About 3% of world population is infected with HCV.