Mechanistic Studies of the Function of Desmosterol Homeostasis in HCV Replication

描述（由申请人提供）：丙型肝炎病毒（HCV）是一种包膜、正链RNA病毒，慢性感染1.8亿人，使他们易患肝硬化、慢性肝炎（肝脏疾病）和肝细胞癌。尽管丙型肝炎流行，但疫苗和广谱抗丙型肝炎药物的开发面临挑战，包括存在多基因型、低疗效和RNA病毒的高突变率特征。虽然最近批准的直接作用病毒抑制剂有望提高治疗效果，但据报道，对这些病毒靶向抑制剂的快速耐药性扩大了对新型抗病毒药物治疗慢性丙型肝炎病毒的需求。针对HCV复制所需的宿主因子的抗病毒药物可能提供几种有吸引力的替代方案

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is an enveloped, plus-stranded RNA virus that chronically infects 180 million people, making them susceptible to cirrhosis, chronic hepatitis (liver disease), and hepatocellular carcinomas. Despite the HCV epidemic, challenges to the development of vaccines and broad-spectrum anti-HCV agents include the existence of multiple genotypes, low efficacy, and the characteristic high mutation rate of RNA viruses. While recently approved directly-acting viral inhibitors are expected to improve treatment efficacy, rapid resistance to these virally-targeted inhibitors has been reported, extending the need for novel antivirals for treatment of chronic HCV. Antivirals that target host factors required for HCV replication may provide an attractive alternative with several advantages over more traditional therapies that target viral factors, including higher barriers to resistance. Understanding the molecular mechanisms underlying productive HCV replication and how HCV manipulates cellular pathways is therefore critical to the design of host-targeted anti-HCV strategies. We recently discovered that HCV causes a specific, ten-fold increase in the abundance of intracellular desmosterol, the penultimate precursor of cholesterol in the Bloch branch of cholesterol biosynthesis, without perturbing the steady-state abundance of cholesterol or other sterols in the pathway. The HCV-induced perturbation of desmosterol homeostasis appears to be critical for HCV replication since inhibition of desmosterol synthesis severely inhibits HCV by decreasing steady-state HCV RNA replication. Here, we propose research designed to elucidate molecular mechanisms by which HCV causes the steady-state accumulation of desmosterol and to understand the functional significance of desmosterol in steady-state HCV genome replication. In Aim 1, we will use immunoprecipitation, immunofluorescence microscopy, and steady state mRNA and protein analyses to investigate whether HCV regulates cellular enzymes involved in late stage cholesterol biosynthesis. In Aim 2, we will use in vitro replication assays and steady state lipid and mRNA analyses to determine the functional significance of desmosterol in the formation, activity, or stability of HCV replicaton complexes (RCs). Last, since steady-state measurements of lipid abundance in cell lysates provide no information regarding the localization of desmosterol in the presence of HCV replication, in Aim 3, we will utilize stimulated Raman spectroscopy imaging methods to examine the subcellular localization of deuterated desmosterol in cells replicating HCV. In particular, we will investigate whether desmosterol accumulates in distinct puncta versus whether it is distributed throughout the cell or remains proximal to the ER. Collectively, these experiments provide unique opportunities to study and validate desmosterol biosynthesis as a potential anti-HCV target and to explore a heretofore uncharacterized mechanism of regulation in cholesterol biosynthesis.