Roles for microRNA-122 in hepatitis C virus RNA amplification

描述（由申请人提供）：HCV仍然是一个重大的健康威胁，有1.7亿人被感染，有效的HCV治疗一直难以捉摸。大多数患者不能解决感染，并成为慢性携带者，增加肝病的风险。事实上，HCV是美国昂贵的肝移植的主要原因。目前还没有针对HCV的疫苗，目前的治疗方法包括利巴韦林和干扰素α，价格昂贵且相对无效。因此，迫切需要新的治疗干预。我们的初步数据显示，在培养的肝细胞中，病毒5'非编码区中的保守区域与肝脏特异性宿主细胞microRNA miR-122相互作用。这种相互作用对于维持细胞内HCV RNA的丰度是必不可少的。在这项提案中，我们将首先研究miR-122对HCV RNA前所未有的上调的调控要求和调控成分。具体而言，我们将使用可转录以产生可复制病毒RNA的HCV cDNA作为工具，测试靶病毒和miR-122 RNA中的序列和结构在miR- 122/HCV RNA复合物形成中的重要性。在目标二中，将检查与HCV RNA相互作用的miR 122-RISC复合物的调节组分。具体而言，将鉴定存在于miR-122-HCV RISC复合物中的特异性argonaute蛋白。具体而言，我们将监测细胞中的HCV RNA水平，其中单个argonaute mRNA已被特定siRNA耗尽，我们将在标记的Ago-RISC复合物的下拉测定中检查HCV RNA的存在。我们还将通过监测病毒RNA是否接受非编码尿苷残基（RNAi途径的标志）来检查HCV RNA是否在特定RISC复合物中被切割。目标三将描述受miR-122影响的病毒生命周期的确切步骤。具体而言，miR-122在调节病毒RNA稳定性、翻译、复制和病毒释放中的作用将在北方和多核糖体分析方法以及新合成的病毒RNA可以用4-硫尿苷残基特异性标记的新系统中进行检查。在最终的目标中，我们将研究阳离子氨基酸转运蛋白CAT-1被miR-122下调的机制，并使用生物信息学方法和一种新的策略来寻找miR-122的其他假定的细胞靶mRNA，通过该策略，生物素化的miR-122可以用作诱饵来纯化细胞靶。最后，我们将使用重组腺相关病毒表达的siRNA敲低大鼠肝脏中的miR-122，并监测对肝功能的影响。这种方法将揭示miR-122是否可以用作新的抗病毒靶点。这些研究的结果将详细说明真核细胞中基因表达的新机制，并可能指出抗HCV治疗的新途径。

DESCRIPTION (provided by applicant): HCV remains a significant health threat, with 170 million people being infected and effective therapies for HCV having been elusive. The majority of patients do not resolve the infection and become chronic carriers with increased risk for liver disease. In fact, HCV is the major cause for expensive liver transplantations in the US. There is no vaccine for HCV, and current treatments, which include ribavirin and interferon alpha are expensive and relatively ineffective. Thus, there is a pressing need for new therapeutic intervention. Our preliminary data showed that a conserved region in the viral 5' noncoding region interacts with a liver-specific host-cell microRNA, miR-122, in cultured liver cells. This interaction is essential to maintain intracellular abundance of HCV RNA. In this proposal, we will first examine the requirements and the regulatory components that govern this unprecedented upregulation of HCV RNA by a miR-122. Specifically, we will test the importance of sequences and structures in the target viral and miR-122 RNA in the formation of miR- 122/HCV RNA complexes using a HCV cDNA that can be transcribed to generate replication-competent viral RNA as a tool. In aim two, the regulatory components of the miR122-RISC complex that interact with the HCV RNA will be examined. In particular, the specific argonaute proteins that reside in miR-122-HCV RISC complexes will be identified. Specifically, we will monitor HCV RNA levels in cells in which individual argonaute mRNAs have been depleted by specific siRNAs and we will examine the presence of HCV RNA in pull-down assays from tagged Ago-RISC complexes. We will also examine whether HCV RNA is cleaved in specific RISC complexes by monitoring whether the viral RNA received nonencoded uridine residues, a signature of the RNAi pathway. Aim three will delineate the exact steps in the viral life cycle that are affected by miR-122. Specifically, roles for miR-122 in modulating viral RNA stability, translation, replication and virus release will be examined in Northern and polysomal profiling approaches and a novel system in which newly synthesized viral RNAs can be specifically labeled with 4-thiouridine residues. In the final aim, we will study the mechanism by which the cationic amino acid transporter CAT-1 is downregulated by miR-122 and search for additional putative cellular target mRNAs for miR-122 using bioinformatics approaches and a novel strategy by which biotinylated miR-122 can be used as a bait to purify cellular targets. Finally, we will knock-down miR-122 in the rat liver using siRNAs expressed from recombinant adeno-associated viruses and monitor effects on liver function. This approach will reveal whether miR-122 can be used a as a novel antiviral target. The outcomes of these studies will detail a novel mechanism of gene expression in eukaryotic cells and may point to new venues of therapies against HCV.