Roles for microRNA-122 in hepatitis C virus RNA amplification

microRNA-122 在丙型肝炎病毒 RNA 扩增中的作用

• 批准号: 7080546
• 负责人: PETER SARNOW
• 金额: $ 30.91万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-15 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7080546
• 关键词: RNA; RNA virus; hepatitis C virus; liver; microRNAs; role; virus RNA

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.

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，并且 HCV 的有效治疗方法一直难以捉摸。大多数患者无法解决感染并成为慢性携带者，肝病风险增加。事实上，丙型肝炎病毒是美国肝移植费用昂贵的主要原因。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。我们的初步数据表明，在培养的肝细胞中，病毒 5' 非编码区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 的丰度至关重要。在本提案中，我们将首先检查 miR-122 对 HCV RNA 进行前所未有的上调的要求和监管组件。具体来说，我们将使用可转录生成具有复制能力的病毒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 miR122-RISC 复合物的调节成分。特别是，将鉴定存在于 miR-122-HCV RISC 复合物中的特定 argonaute 蛋白。具体来说，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，并且我们将在来自标记的 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 途径的特征）来检查 HCV RNA 是否在特定 RISC 复合物中被切割。目标三将描述病毒生命周期中受 miR-122 影响的确切步骤。具体来说，将在 Northern 和多核糖体分析方法以及新系统中检查 miR-122 在调节病毒 RNA 稳定性、翻译、复制和病毒释放中的作用，其中新合成的病毒 RNA 可以用 4-硫尿苷残基特异性标记。在最终目标中，我们将研究 miR-122 下调阳离子氨基酸转运蛋白 CAT-1 的机制，并使用生物信息学方法和生物素化 miR-122 的新策略寻找 miR-122 的其他假定细胞靶 mRNA。 122可用作纯化细胞目标的诱饵。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介绍真核细胞中基因表达的新机制，并可能为丙肝治疗指明新途径。