Mechanistic Studies of the Function of Desmosterol Homeostasis in HCV Replication

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是一种包膜正链RNA病毒，慢性感染1.8亿人，使他们容易患上肝硬变、慢性肝炎(肝病)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发展面临的挑战包括多种基因类型的存在，低效价，以及RNA病毒特有的高突变率。虽然最近批准的直接作用病毒抑制剂有望提高治疗效果，但已有报道对这些病毒靶向抑制剂产生快速耐药性，从而延长了治疗慢性丙型肝炎的新型抗病毒药物的需求。针对丙型肝炎病毒复制所需宿主因素的抗病毒药物可能提供一种有吸引力的替代药物，有几种 与针对病毒因素的更传统疗法相比，包括更高的抗药性障碍，它具有优势。因此，了解丙型肝炎病毒复制的分子机制以及丙型肝炎病毒如何操纵细胞通路，对于设计宿主靶向的抗丙型肝炎病毒策略至关重要。我们最近发现，丙型肝炎病毒导致细胞内桥本甾醇的丰度增加10倍，这是胆固醇生物合成的布洛赫分支中倒数第二个胆固醇前体，而不会扰乱胆固醇或其他甾醇在该途径中的稳定丰度。丙型肝炎病毒诱导的桥粒甾醇稳态的紊乱似乎是丙型肝炎病毒复制的关键，因为抑制桥粒甾醇的合成通过减少稳定状态的丙型肝炎病毒RNA复制而严重抑制丙型肝炎病毒。在这里，我们建议进行研究，旨在阐明丙型肝炎病毒导致桥粒甾醇稳态积累的分子机制，并了解桥粒甾醇在丙型肝炎病毒稳态基因组复制中的功能意义。在目标1中，我们将使用免疫沉淀、免疫荧光显微镜以及稳态信使核糖核酸和蛋白质分析来研究丙型肝炎病毒是否调节参与晚期胆固醇生物合成的细胞酶。在目标2中，我们将使用体外复制分析和稳态脂质和mRNA分析来确定Desmosterol在丙型肝炎病毒复制复合体(RCS)的形成、活性和稳定性中的功能意义。最后，由于对细胞裂解物中脂质丰度的稳态测量没有提供存在丙型肝炎病毒复制时桥粒甾醇定位的信息，在目标3中，我们将利用受激拉曼成像方法来检测重氢桥粒甾醇在复制丙型肝炎病毒的细胞中的亚细胞定位。特别是，我们将调查是否Desmosterol聚集在不同的点状细胞中，还是分布在整个细胞中，还是保持在内质网的近端。总而言之，这些实验提供了独特的机会来研究和验证Desmosterol生物合成作为潜在的抗丙型肝炎病毒靶点，并探索迄今尚不清楚的胆固醇生物合成调节机制。

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.