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: 8040024
• 负责人: PETER SARNOW
• 金额: $ 39.09万
• 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 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8040024
• 关键词: Address; Adenosine; Affect; Anatomy; Animals; Antiviral Agents; Antiviral Therapy; Binding; Binding Sites; Biochemical; Cells; Cholesterol; Cholesterol Homeostasis; Chronic; Combined Modality Therapy; Complex; Confocal Microscopy; Cultured Cells; Dependence; Double-Stranded RNA; Down-Regulation; Drops; Elements; Enzymes; Eukaryotic Cell; Gene Expression; Genes; Genetic; Genome; Goals; Guanosine; HCV Vaccine; Healed; Health; Hepatitis C virus; Hepatocyte; Immune response; Individual; Infection; Interferons; Internal Ribosome Entry Site; Intervention; Lead; Life Cycle Stages; Liver; Liver diseases; Maintenance; Mediating; Messenger RNA; MicroRNAs; Modification; Monitor; Nucleotides; Occupations; Outcome Study; Pan Genus; Patients; Poly A; Polyadenylation; Primary carcinoma of the liver cells; Protein Isoforms; Proteins; RNA; RNA Degradation; RNA amplification; Recruitment Activity; Resistance; Ribavirin; Ribonucleases; Role; Science; Sequence Analysis; Serum; Site; Small Interfering RNA; Staging; Structure; Substrate Specificity; Testing; Transferase; Tretinoin; United States; Up-Regulation; Viral; Viral Genome; Viral Load result; Virus; cellular targeting; cholesterol biosynthesis; effective therapy; fatty acid metabolism; genome-wide; healing; liver transplantation; locked nucleic acid; mimetics; mutant; novel; nuclease; research study; sensor; viral RNA

DESCRIPTION (provided by applicant): An estimated 170 million people worldwide and 4 million people in the United States are infected with hepatitis C virus (HCV). The majorities of patients do not resolve the infection and develop chronic infections that often lead to end-stage liver disease and hepatocellular carcinoma. Current treatment is limited to a combination therapy of ribavirin and interferon 1. This therapy is expensive and ineffective in 50% of infected individuals. Thus, there is an urgent need to identify viral or cellular molecules that can be used as novel targets in antiviral therapy. It was discovered that HCV binds two molecules of a liver-specific microRNA, miR-122, resulting in a novel, unprecedented upregulation of the viral genome. Sequestration of miR-122 in HCV-infected cultured cells or in livers of infected chimpanzees leads to a dramatic loss of infectious virus without emergence of resistant virus. Therefore, the dependence of HCV on miR-122 presents an Achilles heel of the virus that can be explored for antiviral intervention. This application proposes to study the roles for miR-122 in the viral life cycle and in cholesterol biosynthesis using a novel class of antisense molecules, locked nucleic acids (LNAs) that can easily be delivered to the liver in animals where it sequesters miR-122 in an inactive small duplex RNA. In particular, the first aim will characterize the RNA-RNA interactions in the miR-122/HCV complex, using genetic and biochemical approaches. Aim 2 will test the hypothesis that miR-122 protects the 5' end sequences of the HCV RNA from degradation by ribonucleases or RNA modification enzymes, or aids in the avoidance of activation of double-stranded RNA sensors such as the retinoic acid inducible gene I. These studies will be performed in specific and genome-wide siRNA-mediated gene knockdown experiments. Aim 3 proposes to examine roles for the known isoforms of miR-122 that contain extra 3' terminal adenosine or guanosine residues, on HCV RNA abundance. Deep sequencing analysis and gene knockdown of suspected nucleotidyl transferases will aid in this analysis. The final aim will characterize in detail the mechanism by which miR-122 regulates the expression of Insig1, the major negative regulator of cholesterol and fatty acid metabolism in the liver. In particular, the miR-122-mediated down-regulation of a distinct polyadenylation/cleavage site in a specific Insig1 isoform mRNAs will be examined. Overall, this application will address fundamental aspects about the functions of miR-122 in the HCV life cycle and cholesterol metabolism. The outcomes from these studies will detail novel mechanisms of gene expression mediated by microRNAs in eukaryotic cells and will point to new venues for antiviral therapies. PUBLIC HEALTH RELEVANCE: An estimated 170 million people worldwide and 4 million in the United States are infected with hepatitis C virus (HCV). The majority of patients do not resolve the infection and become chronic carriers, ultimately needing expensive liver transplants. There is no vaccine for HCV, and current treatments, which include ribavirin and interferon 1, are expensive and relatively ineffective. It was discovered that HCV binds two molecules of a liver- specific microRNA, miR-122, resulting in a novel, unprecedented upregulation of the viral genome. This proposal explores the mechanisms by which miR-122 protects HCV RNA in the liver. The dependence of HCV on miR-122 presents an Achilles heel of the virus that can be used for antiviral intervention. We will study the roles for miR-122 in the viral life cycle and in cholesterol biosynthesis using a novel class of antisense molecules (locked nucleic acids) that can easily bind and inactivate miR-122 in the liver of animals. This is a highly significant approach, because LNA-mediated sequestration of miR-122 in the liver of HCV-infected chimpanzees resulted in a 2.5 fold drop in viral load without any emergence of resistant virus (Lanford et al. 2010. Science: 327:198-201).

描述（由申请人提供）：估计全世界有 1.7 亿人、美国有 400 万人感染丙型肝炎病毒 (HCV)。大多数患者无法解决感染并发展为慢性感染，通常导致终末期肝病和肝细胞癌。目前的治疗仅限于利巴韦林和干扰素 1 的联合疗法。这种疗法价格昂贵，而且对 50% 的感染者无效。因此，迫切需要鉴定可用作抗病毒治疗新靶点的病毒或细胞分子。研究发现，HCV 与肝脏特异性 microRNA（miR-122）的两个分子结合，导致病毒基因组出现新颖的、前所未有的上调。 miR-122 被隔离在 HCV 感染的培养细胞或受感染黑猩猩的肝脏中，导致感染性病毒急剧减少，而不会出现耐药病毒。因此，HCV 对 miR-122 的依赖性是该病毒的致命弱点，可用于抗病毒干预。本申请拟使用一类新型反义分子、锁定核酸 (LNA) 来研究 miR-122 在病毒生命周期和胆固醇生物合成中的作用，该分子可以轻松递送至动物肝脏，在那里隔离 miR-122在无活性的小双链体 RNA 中。特别是，第一个目标将使用遗传和生化方法来表征 miR-122/HCV 复合物中 RNA-RNA 相互作用的特征。目标 2 将检验以下假设：miR-122 保护 HCV RNA 的 5' 末端序列免遭核糖核酸酶或 RNA 修饰酶的降解，或有助于避免双链 RNA 传感器（例如视黄酸诱导基因 I）的激活这些研究将在特定的全基因组 siRNA 介导的基因敲除实验中进行。目标 3 提议检查含有额外 3' 末端腺苷或鸟苷残基的已知 miR-122 亚型对 HCV RNA 丰度的作用。对可疑核苷酸转移酶的深度测序分析和基因敲除将有助于此分析。最终目标将详细描述 miR-122 调节 Insig1 表达的机制，Insig1 是肝脏中胆固醇和脂肪酸代谢的主要负调节因子。特别是，将检查 miR-122 介导的特定 Insig1 同工型 mRNA 中不同聚腺苷酸化/切割位点的下调。总体而言，该应用将解决有关 miR-122 在 HCV 生命周期和胆固醇代谢中功能的基本问题。这些研究的结果将详细说明真核细胞中 microRNA 介导的基因表达的新机制，并将为抗病毒治疗指明新的途径。 公共卫生相关性：估计全世界有 1.7 亿人、美国有 400 万人感染丙型肝炎病毒 (HCV)。大多数患者无法解决感染并成为慢性携带者，最终需要昂贵的肝移植。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 1）价格昂贵且相对无效。研究发现，HCV 与肝脏特异性 microRNA（miR-122）的两个分子结合，导致病毒基因组出现新的、前所未有的上调。该提案探讨了 miR-122 保护肝脏中 HCV RNA 的机制。 HCV 对 miR-122 的依赖性是该病毒可用于抗病毒干预的致命弱点。我们将使用一类新型反义分子（锁定核酸）来研究 miR-122 在病毒生命周期和胆固醇生物合成中的作用，该反义分子可以轻松结合并灭活动物肝脏中的 miR-122。这是一种非常重要的方法，因为 LNA 介导的 miR-122 在 HCV 感染的黑猩猩肝脏中的隔离导致病毒载量下降 2.5 倍，而没有出现任何耐药病毒（Lanford 等人，2010 年。 Science：327： 198-201）。