HCV biology and inhibition visualized at the single molecule level

描述（由申请人提供）：丙型肝炎病毒（HCV）是黄病毒科的一种正义单链RNA病毒，在美国造成的死亡人数超过艾滋病毒。丙型肝炎病毒感染是慢性肝炎、肝硬化和肝细胞癌的主要原因，目前全球约有1.8亿人感染丙型肝炎病毒，其中约400万人居住在美国。目前还没有针对丙肝病毒的保护性疫苗。目前的治疗标准是聚乙二醇化干扰素和利巴韦林的联合治疗，但效果有限，副作用严重。最近FDA批准的蛋白酶抑制剂Telaprevir和Boceprevir必须与这种标准治疗一起使用，以实现持续的病毒清除，因此即使是最新的治疗方法仍然存在严重的副作用。我们的合作者Peter Sarnow（斯坦福大学）最近发现了microRNA (miRNA) miR-122在HCV复制中的显著保护作用，为抑制内源性miRNA的抗mirs作为一类新型HCV药物铺平了道路。此外，我们的合作者SomaGenics Inc.的Brian Johnston最近开发了几种小干扰RNA （sirna）以及短小发卡RNA (sshRNAs)，它们在细胞培养中介导有效的RNA干扰（RNAi）来对抗HCV。本R21提案的主要目的是解剖潜在的细胞内

DESCRIPTION (provided by applicant): The hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus of the Flaviviridae family that causes more deaths in the US than HIV. Infection with HCV is the leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, and currently affects ~180 million people worldwide, ~4 million of whom reside in the US. No protective vaccines exist against HCV. The current standard of care is a combination of pegylated interferon and ribavirin, with limited effectiveness and severe side effects. The recently FDA- approved protease inhibitors Telaprevir and Boceprevir have to be administered with this standard treatment to achieve sustained viral clearance so that even the newest therapy is still associated with serious side effects. The recent discovery by our collaborator Peter Sarnow (Stanford University) of a remarkable protective role for microRNA (miRNA) miR-122 in HCV replication has paved the way for anti-miRs, which inhibit endogenous miRNAs, as a novel class of HCV drugs. In addition, our collaborator Brian Johnston from SomaGenics Inc. recently developed several small interfering RNAs (siRNAs) as well as short small hairpin RNAs (sshRNAs) that mediate efficient RNA interference (RNAi) against HCV in cell culture. The primary objective of this R21 proposal is to dissect the underlying intracellular mechanisms of these promising small-RNA anti-HCV drugs, with the long-term goal of further improving their efficacy. To this end, we will bring to bear the powerful capabilities of Single molecule, High-Resolution Localization and Counting (iSHiRLoC), an innovative probe concept we recently developed for detecting the trafficking and assembly of single miRNA molecules inside live cultured cells. iSHiRLoC entails single particle tracking of physiologically relevant lw numbers of microinjected, inconspicuously fluorophore-labeled RNAs to record their spatiotemporal distribution and count their stoichiometry upon RNA:protein (RNP) complex assembly. We propose to expand iSHiRLoC to two colors and study HCV biology and inhibition at an unprecedented single molecule level, by pursuing the following three Specific Aims: (i) We will probe the mechanism of miR-122-mediated intracellular protection of HCV RNA by using two-color iSHiRLoC in cultured, miR-122-free HepG2 hepatocytes and comparing the spatiotemporal distributions and assembly stoichiometries of Cy5-labeled miR-122, microinjected in conjunction with either Cy3-labeled subgenomic HCV RNA or Cy3-labeled AldoA target mRNA. (ii) We will probe the mechanism of siRNA- and sshRNA-mediated intracellular RNA interference against HCV by applying two-color iSHiRLoC to siRNAs and sshRNAs already validated against HCV in hepatocytes by SomaGenics Inc. (iii) We will observe the "tug-of-war" between miR-122-mediated protection of and RNAi against HCV RNA by performing a precise intracellular "titration" of Cy3-labeled HCV RNA with varying amounts of unlabeled miR-122 in the presence of our most effective Cy5-labeled siRNA/shRNA to directly observe the evolution of the HCV RNA spatiotemporal distribution as the "tug-of-war" between the two small RNAs plays out.