Roles for microRNA-122 in hepatitis C virus RNA amplification

HCV仍然是一个重大的健康威胁，有1.7亿人被感染，有效的治疗方法 HCV一直难以捉摸。大多数患者不能解决感染，成为慢性携带者 肝脏疾病的风险增加。事实上，丙型肝炎病毒是昂贵的肝移植的主要原因， 美方目前还没有针对HCV的疫苗，目前的治疗方法包括利巴韦林和干扰素α。 昂贵且相对无效。因此，迫切需要新的治疗干预。我们 初步数据显示，病毒5'非编码区中的保守区域与肝特异性 宿主细胞microRNA，miR-122，在培养的肝细胞。这种相互作用对于维持细胞内 HCV RNA的丰度。在本建议中，我们将首先研究要求和监管 这是一种由miR-122控制HCV RNA前所未有的上调的组分。具体来说，我们将 测试靶病毒和miR-122 RNA中的序列和结构在miR-122形成中的重要性。 122/HCV RNA复合物，其使用可被转录以产生复制能力的病毒的HCV cDNA RNA是一种工具。在目的二中，miR 122-RISC复合物中与miR-122相互作用的调节组分被称为miR-122-RISC。 将检查HCV RNA。特别是，存在于miR-122-HCV RISC中的特异性argonaute蛋白， 复合体将被识别。具体地说，我们将监测细胞中的HCV RNA水平， argonaute mRNAs已经被特定的siRNA耗尽，我们将在HCV RNA的表达水平上检测HCV RNA的存在。 从标记的Ago-RISC复合物中提取的样品。我们还将检查HCV RNA是否在 特异性RISC复合物，通过监测病毒RNA是否接受非编码尿苷残基， RNAi途径的特征。第三个目标将描述病毒生命周期中受影响的确切步骤 miR-122具体而言，miR-122在调节病毒RNA稳定性、翻译、复制和病毒复制中的作用是通过调节miR-122的表达来实现的。 释放将在北方和多聚体分析方法和一个新的系统，其中新的 合成的病毒RNA可以用4-硫代尿苷残基特异性标记。在最终的目标，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， 使用生物信息学方法和一种新的 生物素化的miR-122可用作诱饵以纯化细胞靶标的策略。最后，我们会敲门- 使用从重组腺相关病毒表达的siRNA下调大鼠肝脏中的miR-122， 监测对肝功能的影响。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一种新的基因表达机制， 真核细胞，并可能指向新的途径治疗丙型肝炎病毒。

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 cDNAthat 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 asays 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.