HCV biology and inhibition visualized at the single molecule level

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是黄病毒科家族的一种阳性单链RNA病毒，在美国导致的死亡人数超过艾滋病毒。丙型肝炎病毒感染是慢性肝炎、肝硬变和肝细胞癌的主要原因，目前影响着全球约1.8亿人，其中约400万人居住在美国。目前还没有针对丙型肝炎病毒的保护性疫苗。目前的治疗标准是聚乙二醇化干扰素和利巴韦林的组合，疗效有限，副作用严重。最近FDA批准的蛋白酶抑制剂telaprevir和bocprevir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与严重的副作用有关。我们的合作者Peter Sarnow(Stanford University)最近发现，microRNA(MiRNA)miR-122在丙型肝炎病毒复制中具有显著的保护作用，这为抗miR(抑制内源性miRNAs)作为一类新型丙型肝炎药物铺平了道路。此外，我们的合作者SomaGenics Inc.的Brian Johnston最近开发了几个小干扰RNA(SiRNAs)和短小发夹RNA(SshRNAs)，它们在细胞培养中介导对丙型肝炎病毒的有效RNA干扰(RNAi)。该R21提案的主要目标是剖析潜在的细胞内 这些有希望的小RNA抗丙型肝炎药物的作用机制，长期目标是进一步提高它们的疗效。为此，我们将利用我们最近开发的创新探针概念-单分子高分辨率定位和计数(ISHiRLoC)-来检测活培养细胞内单个miRNA分子的运输和组装。ISHiRLoC需要对显微注射的、不明显的荧光团标记的RNA的生理相关LW数量进行单粒子跟踪，以记录它们的时空分布，并在RNA：蛋白质(RNP)复合体组装上计算它们的化学计量比。我们建议将iSHiRLoC扩展到两种颜色，并通过追求以下三个特定目标在前所未有的单分子水平上研究丙型肝炎病毒的生物学和抑制作用：(I)我们将通过在培养的、不含miR-122的HepG2肝细胞中使用双色iSHiRLoC，并比较显微注射Cy5标记的miR-122与Cy3标记的亚基因组丙型肝炎病毒RNA或Cy3标记的AldoA靶mRNA一起显微注射，来探讨miR-122介导的丙型肝炎病毒RNA的细胞内保护机制，并比较Cy5标记的miR-122与Cy3标记的丙型肝炎病毒RNA或Cy3标记的AldoA靶mRNA一起显微注射的时空分布和组装计量学。(Ii)我们将通过将双色iSHiRLoC应用于SomaGenics Inc.已经验证过的针对肝细胞中丙型肝炎病毒的siRNAs和sshRNAs，来探索siRNA和sshRNA介导的细胞内RNA干扰抗丙型肝炎病毒的机制。(Iii)我们将通过在我们最有效的Cy5标记的siRNA/shRNA的存在下，通过精确的细胞内“滴定”不同数量的未标记的miR-122来观察miR-122介导的对丙型肝炎病毒RNA的保护和RNAi之间的“拉锯战”，以直接观察丙型肝炎病毒RNA时空分布的演变。

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.