Roles for microRNA-122 in hepatitis C virus RNA amplification

描述（由申请人提供）：丙型肝炎病毒仍然是一个重大的健康威胁，有1.7亿人感染，丙型肝炎病毒的有效治疗方法尚未找到。大多数患者没有解决感染，成为慢性携带者，增加了肝脏疾病的风险。事实上，丙型肝炎病毒是美国肝脏移植费用昂贵的主要原因。目前还没有针对丙肝病毒的疫苗，目前的治疗方法，包括利巴韦林和干扰素，都是昂贵且相对无效的。因此，迫切需要新的治疗干预措施。我们的初步数据显示，在培养的肝细胞中，病毒5'非编码区的一个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的丰度至关重要。在本提案中，我们将首先研究miR-122对HCV RNA的这种前所未有的上调的要求和调控成分。具体来说，我们将测试目标病毒和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，使用HCV cDNA作为工具，可以转录生成复制能力强的病毒RNA。在目标二中，将研究miR122-RISC复合体与HCV RNA相互作用的调控成分。特别是，将鉴定驻留在miR-122-HCV RISC复合体中的特异性argonaute蛋白。具体来说，我们将监测单个argonaute mrna被特定sirna耗尽的细胞中的HCV RNA水平，我们将在标记Ago-RISC复合物的下拉试验中检查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途径的一个标志）来检测HCV RNA是否在特定的RISC复合体中被切割。目标三将描述受miR-122影响的病毒生命周期中的确切步骤。具体来说，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Northern和多染色体分析方法中进行研究，并在一个新合成的病毒RNA可以用4-硫脲残基特异性标记的新系统中进行研究。在最后的目标中，我们将研究阳离子氨基酸转运体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的其他可能的细胞靶mrna，通过这种策略，生物素化的miR-122可以用作纯化细胞靶标的诱饵。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，并监测其对肝功能的影响。这种方法将揭示miR-122是否可以作为一种新的抗病毒靶点。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治疗的新领域。

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.