Roles for microRNA-122 in hepatitis C virus RNA amplification

丙型肝炎病毒仍然是一个重大的健康威胁，有1.7亿人被感染，并有有效的治疗方法 丙型肝炎病毒一直难以捉摸。大多数患者不能消除感染，成为慢性携带者 会增加患肝病的风险。事实上，丙型肝炎病毒是我国昂贵的肝移植手术的主要原因。 美国。目前还没有丙型肝炎病毒的疫苗，目前的治疗方法包括利巴韦林和干扰素。 都很昂贵，而且效果相对较差。因此，迫切需要新的治疗干预措施。我们的 初步数据显示，病毒5‘非编码区的保守区与肝脏特异的 宿主细胞microRNA，miR-122，在培养的肝细胞中。这种相互作用对于维持细胞内的 丙型肝炎病毒核糖核酸含量丰富。在这项建议中，我们将首先审查要求和监管 管理这一史无前例的丙型肝炎病毒RNA上调的miR-122的组件。具体来说，我们将 检测靶病毒和miR-122 RNA中的序列和结构在miR-122形成中的重要性 122/丙型肝炎病毒RNA复合体，使用可转录的丙型肝炎病毒cDNA产生复制能力强的病毒 RNA作为一种工具。在目标二中，miR122-RISC复合体的调节组件与 将检查丙型肝炎病毒核糖核酸。特别是，驻留在miR-122-丙型肝炎病毒RISC中的特定ArgAerte蛋白 复合体将被识别出来。具体地说，我们将监测细胞中的丙型肝炎病毒RNA水平 ArgAerte的mRNAs已经被特定的siRNAs耗尽，我们将检查在 来自标记的AGO-RISC复合体的下拉ASS。我们还将检查丙型肝炎病毒RNA是否被切割成 通过监测病毒RNA是否接收到非编码尿苷残基，产生特异性RISC复合体 RNAi途径的信号。目标三将描述病毒生命周期中受影响的确切步骤 由MIR-122制造。具体地说，miR-122在调节病毒RNA稳定性、翻译、复制和病毒方面的作用 释放将通过Northern和多体谱方法以及一种新的系统进行检查，在该系统中 合成的病毒RNA可以被4-硫代尿苷残基特异性标记。在最终目标中，我们将研究 MiR-122和Search下调阳离子氨基酸转运体CAT-1的机制 使用生物信息学方法和一种新的方法为miR-122额外的假定细胞靶向RNA 生物素化miR-122可用作诱饵以纯化细胞靶标的策略。最后，我们要敲门- 使用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是否可以作为一种小说使用 抗病毒目标。这些研究的结果将详细说明一种新的基因表达机制 并可能为抗丙型肝炎病毒的治疗开辟新的途径。

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.