CELLULAR GENES THAT CONTROL HCV REPLICATION

描述(由申请人提供)：这项建议的主要目标是确定调控丙型肝炎病毒(丙型肝炎病毒)在肝细胞中复制的细胞基因。这项应用是基于最近描述的肝脏基因表达谱，该谱表征了实验感染丙型肝炎病毒的黑猩猩感染过程中的不同时间点。确定了三组基因的表达与(A)感染的开始和持续时间，(B)感染的程度，和(C)感染的缓解有关。在这些研究中，几个与脂代谢有关的基因被证明与感染的程度有关。为验证这些观察结果的相关性而设计的实验表明，刺激或抑制胆固醇和脂肪酸生物合成的药物分别增强或抑制了亚基因组丙型肝炎复制子在Huh-7细胞中的复制。在目前的应用中，包含亚基因组和全长丙型肝炎病毒复制子的Huh-7细胞将被用来确定在感染的黑猩猩中发现的任何基因是否可以控制丙型肝炎病毒在肝细胞中的复制。这些基因在Huh-7细胞中的表达将被RNA干扰抑制或通过转基因增强，这些操作对丙型肝炎病毒复制的影响将被评估。特定的目标1-3将检查在黑猩猩中识别的三组肝基因是否需要维持基础水平的丙型肝炎病毒复制(目标1)，增强基础水平以上的丙型肝炎病毒复制(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，将确定调控丙型肝炎病毒复制的细胞胆固醇和脂肪酸生物合成的特定步骤(S)，并将评估细胞脂代谢对丙型肝炎病毒蛋白在细胞内定位和相互作用的影响。通过阐明控制丙型肝炎病毒复制的细胞调控机制，本申请中描述的研究可能确定治疗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.