CELLULAR GENES THAT CONTROL HCV REPLICATION

描述（由申请人提供）：本申请的主要目的是鉴定在肝细胞中调节丙型肝炎病毒（HCV）复制的细胞基因。该应用程序基于最近描述的肝基因表达谱，该谱表征了实验感染的黑猩猩在HCV感染过程中的不同时间点。鉴定出三组基因，它们的表达与(a)感染的发生和持续时间，(b)感染的程度和(c)感染的消退相关。在这些研究中，一些参与脂质代谢的基因被证明与感染的程度有关。旨在验证这些观察结果相关性的实验表明，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Huh-7细胞中亚基因组HCV复制子的复制。在目前的应用中，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黑猩猩中发现的任何基因是否可以控制HCV在肝细胞中的复制。这些基因在Huh-7细胞中的表达将被RNA干扰抑制或通过转染增强，这些操作对HCV复制的影响将被评估。具体目标1-3将检查在黑猩猩中发现的三组肝脏基因是否需要维持HCV复制的基础水平（目标1），以增强HCV复制的基础水平（目标2）；或介导干扰素的抗病毒作用（目的3）。此外，在Aim 4中，将确定调节HCV复制的细胞胆固醇和脂肪酸生物合成的具体步骤，并评估细胞脂质代谢对HCV蛋白细胞内定位和相互作用的影响。通过阐明控制HCV复制的细胞调控机制，本应用程序中描述的研究可能确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.