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

基本信息

批准号：
7570062
负责人：
PETER SARNOW
金额：
$ 29.6万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
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/7570062
关键词：
Affect Antiviral Agents Antiviral Therapy Basic Amino Acid Transport Systems Binding Sites Biochemical Bioinformatics Biological Assay Biotinylation Cells Chronic Cleaved cell Collaborations Complementary DNA Complex Cultured Cells Data Detection Down-Regulation Elements Eukaryotic Cell Gene Expression Genes HCV Vaccine Healed Health Hepatitis C virus Hepatocyte Individual Infection Interferon-alpha Internal Ribosome Entry Site Label Lead Life Cycle Stages Liver Liver diseases Maps Mediating MicroRNAs Monitor Mutagenesis Nucleotides Outcome Study Pathogenesis Pathway interactions Patients Proteins RNA RNA Interference RNA Stability RNA amplification RNA-Induced Silencing Complex Rattus Reagent Recombinant adeno-associated virus (rAAV)Recruitment Activity Research Personnel Ribavirin Risk Role Small Interfering RNA Streptavidin Structure System Testing Thiouridine Translations Up-Regulation Uridine Viral Viral Genome Virion Virus Virus Replication cellular targeting effective therapy healing knock-down liver function liver transplantation novel novel strategies novel therapeutic intervention nuclease programs promoter reconstitution stem tool viral RNA

项目摘要

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.

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。大多数患者无法解决感染并成为慢性携带者患肝病的风险增加。事实上，HCV 是导致肝移植费用昂贵的主要原因。美国。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，病毒 5' 非编码区的保守区域与肝脏特异性的相互作用培养的肝细胞中的宿主细胞 microRNA，miR-122。这种相互作用对于维持细胞内 HCV RNA 丰度。在本提案中，我们将首先审查要求和监管 miR-122 控制 HCV RNA 前所未有的上调。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- 形成中的重要性 122/HCV RNA 复合物使用 HCV cDNA，可转录生成具有复制能力的病毒 RNA作为工具。在目标二中，miR122-RISC 复合物的调控组件与将检查 HCV RNA。特别是，存在于 miR-122-HCV RISC 中的特定 argauute 蛋白复合物将被识别。具体来说，我们将监测个体细胞中的 HCV RNA 水平 argonaute mRNA 已被特定的 siRNA 耗尽，我们将检查 HCV RNA 的存在从标记的 Ago-RISC 复合物中进行下拉分析。我们还将检查 HCV RNA 是否被切割通过监测病毒RNA是否接受非编码的尿苷残基来识别特定的RISC复合物， RNAi 途径的特征。目标三将描述病毒生命周期中受影响的确切步骤通过 miR-122。具体来说，miR-122 在调节病毒 RNA 稳定性、翻译、复制和病毒中的作用释放将在北方和多核糖体分析方法和一个新的系统中进行检查，其中新合成的病毒RNA可以用4-硫尿苷残基特异性标记。为了最终的目标，我们将学习 miR-122下调阳离子氨基酸转运蛋白CAT-1的机制及搜索使用生物信息学方法和新颖的方法寻找 miR-122 的其他假定细胞靶 mRNA 生物素化的 miR-122 可以用作纯化细胞靶标的诱饵。最后，我们会敲—— 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 是否可以用作新型药物抗病毒靶点。这些研究的结果将详细介绍基因表达的新机制真核细胞，并可能为丙型肝炎治疗提供新的途径。