CELLULAR GENES THAT CONTROL HCV REPLICATION

描述（由申请人提供）：本提案的主要目的是鉴定调节肝细胞中丙型肝炎病毒（HCV）复制的细胞基因。 该应用是基于最近描述的肝脏基因表达谱，其特征在于实验感染的黑猩猩中HCV感染过程中的不同时间点。 鉴定了三组基因，其表达与（a）感染的发作和持续时间，（B）感染的程度，和（c）感染的消退相关。 在这些研究中，参与脂质代谢的几个基因被证明与感染的程度有关。 旨在验证这些观察结果的相关性的实验显示，Huh-7细胞中亚基因组HCV复制子的复制分别被刺激或抑制胆固醇和脂肪酸生物合成的药物增强或抑制。 在本申请中，含有亚基因组和全长HCV复制子的Huh-7细胞将用于确定在感染的黑猩猩中鉴定的任何基因是否可以控制肝细胞中的HCV复制。 这些基因在Huh-7细胞中的表达将通过RNA干扰抑制或通过转染增强，并且将评估这些操作对HCV复制的影响。 具体目标1-3将检查在黑猩猩中鉴定的三组肝脏基因是否需要维持HCV复制的基础水平（目标1），增强HCV复制高于基础水平（目标2）;或介导干扰素的抗病毒作用（目标3）。 此外，在目标4中，将确定细胞胆固醇和脂肪酸生物合成中调节HCV复制的特定步骤，并评估细胞脂质代谢对HCV蛋白质细胞内定位和相互作用的影响。 通过阐明控制HCV复制的细胞调节机制，本申请中描述的研究可以鉴定治疗性抗病毒干预的新靶点。

DESCRIPTION (provided by applicant): The primary objective of this proposal is to identify cellular genes that regulate hepatitis C virus (HCV) replication in the hepatocyte. The application is based on recently described liver gene expression profiles that characterize different time points during the course of HCV infection in experimentally infected chimpanzees. Three groups of genes were identified whose expression correlated with (a) the onset and duration of infection, (b) the magnitude of infection, and (c) the resolution of infection. Several genes involved in lipid metabolism were shown to be associated with the magnitude of infection in those studies. Experiments designed to validate the relevance of those observations revealed that replication of a subgenomic HCV replicon in Huh-7 cells was enhanced or suppressed by drugs that stimulate or inhibit cholesterol and fatty acid biosynthesis, respectively. In the current application, Huh-7 cells containing subgenomic and full length HCV-replicons will be used to determine if any of the genes identified in the infected chimpanzees can control HCV replication in hepatocytes. Expression of these genes in Huh-7 cells will be inhibited by RNA interference or enhanced by transfection, and the effect of those manipulations on HCV replication will be assessed. Specific Aims 1-3 will examine whether the three groups of liver genes identified in the chimpanzees are required either to maintain basal levels of HCV replication (Aim 1), to enhance HCV replication above basal levels (Aim 2); or to mediate the antiviral effects of interferon (Aim 3). In addition, in Aim 4, the specific step(s) in cellular cholesterol and fatty acid biosynthesis that regulate HCV replication will be identified, and the impact of cellular lipid metabolism on the intracellular localization and interactions of HCV proteins will be assessed. By elucidating the cellular regulatory mechanisms that control HCV replication, the studies described in this application may identify new targets for therapeutic antiviral intervention.