Roles for microRNA-122 in hepatitis C virus RNA amplification

描述(申请人提供)：丙型肝炎病毒仍然是一个严重的健康威胁，有1.7亿人被感染，丙型肝炎病毒的有效治疗一直难以找到。大多数患者不能消除感染，成为慢性携带者，患肝病的风险增加。事实上，丙型肝炎病毒是美国昂贵的肝脏移植手术的主要原因。目前还没有针对丙型肝炎的疫苗，目前的治疗方法，包括利巴韦林和α干扰素，都很昂贵，而且相对无效。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‘非编码区的保守区与肝脏特异的宿主细胞microRNA miR-122相互作用。这种相互作用对于维持细胞内丙型肝炎病毒RNA的丰度至关重要。在这项提案中，我们将首先研究管理这一史无前例的通过miR-122上调丙型肝炎病毒RNA的要求和监管组成部分。具体地说，我们将测试目标病毒和miR-122 RNA中的序列和结构在形成miR-122/HCVRNA复合体中的重要性，该复合体可以被转录以产生具有复制能力的病毒RNA作为工具。在目标二，将检查miR122-RISC复合体与丙型肝炎病毒RNA相互作用的调节成分。特别是，驻留在miR-122-丙型肝炎病毒RISC复合体中的特定Argavite蛋白将被鉴定。具体地说，我们将监测单个Argavite mRNAs被特定siRNAs耗尽的细胞中的丙型肝炎病毒RNA水平，并将检查标记的AGO-RISC复合体的下拉分析中是否存在丙型肝炎病毒RNA。我们还将通过监测病毒RNA是否收到非编码尿苷残基来检查丙型肝炎病毒RNA是否在特定的RISC复合体中被切割，尿苷残基是RNAi途径的特征。目标三将描绘病毒生命周期中受miR-122影响的确切步骤。具体地说，miR-122在调节病毒RNA稳定性、翻译、复制和病毒释放方面的作用将在Northern和多体谱方法以及一种新的系统中进行研究，在该系统中，新合成的病毒RNA可以被4-硫代尿苷残基特异性标记。最终，我们将研究miR-122下调阳离子氨基酸转运体CAT-1的机制，并利用生物信息学方法和一种新的策略，即生物素化的miR-122可以作为诱饵来纯化细胞靶标，寻找额外的miR-122的细胞靶标。最后，我们将使用重组腺相关病毒表达的siRNAs在大鼠肝脏中击倒miR-122，并监测对肝功能的影响。这一方法将揭示miR-122是否可以被用作新的抗病毒靶点。这些研究的结果将详细说明在真核细胞中基因表达的新机制，并可能指出治疗丙型肝炎病毒的新途径。

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.