Micro-RNA 122 and Chronic Hepatitis C

Micro-RNA 122 和慢性丙型肝炎

• 批准号: 8258235
• 负责人: Stanley M. Lemon
• 金额: $ 36.84万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-15 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8258235
• 关键词: Adopted; Affinity; Antiviral Agents; Antiviral Therapy; Base Pairing; Binding; Biological Assay; Cell-Free System; Cells; Cessation of life; Chronic Hepatitis C; Cirrhosis; Complex; Data; Dependence; Development; Disease; Drug usage; Fluorescence Resonance Energy Transfer; Genetic; Genome; HIV; Hepatitis C virus; Immunoprecipitation; Infectious hepatitides; Injection of therapeutic agent; Internal Ribosome Entry Site; Laboratories; Life; Life Cycle Stages; Liver; Malignant Neoplasms; Membrane; MicroRNAs; Morbidity - disease rate; Pan Genus; Pathogenesis; Phase; Phosphodiesterase I; Play; Positioning Attribute; Production; Proteins; Proteomics; Publishing; RNA; RNA Degradation; RNA Interference; RNA Stability; RNA chemical synthesis; Recording of previous events; Response Elements; Ribonucleoproteins; Role; Series; Testing; Therapeutic Effect; Translations; Untranslated RNA; Vesicle; Viral; Viral Proteins; Virus; Virus Replication; Work; cellular imaging; human disease; insight; knock-down; mortality; novel; protein expression; public health relevance; replicase; therapeutic target; viral RNA

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is a major cause of liver-specific morbidity and mortality worldwide, resulting in >350,000 deaths annually due to cirrhosis and cancer with high rates of disease among those co-infected with human immunodeficiency virus (HIV) or having a history of injection drug use. microRNA-122 (miR-122), an abundant, liver-specific miRNA is essential for replication of infectious HCV and thus plays a novel role in the pathogenesis of chronic hepatitis C. Recent chimpanzee studies have validated miR-122 silencing as an effective antiviral strategy, yet how miR-122 promotes HCV replication is not well understood. Recent work shows that miR-122 promotes viral translation through direct interactions with a miRNA response element at the 5' end of (+)-strand RNA, while new preliminary data indicate that it also acts to stabilize the RNA. This project will investigate the hypothesis that miR-122, in association with Ago and possibly other cellular proteins, forms a ribonucleoprotein complex (the "miRNA-associated stabilization complex", or MASC) at the 5' end of (+)-strand HCV RNA. The MASC complex has several proposed functions: (i) it protects (+)-strand RNA within the cytoplasmic compartment from degradation, thereby increasing RNA available for translation and assembly into membrane-bound replicase complexes; (ii) it may directly enhance the translational activity of HCV RNA; and (iii), it may promote new viral RNA synthesis by facilitating macroassembly of replicase complexes and/or de novo initiation of RNA synthesis. A series of interrelated aims will rigorously test these hypotheses, determine RNA and protein components of the MASC, and further characterize the novel functions of this unique ribonucleoprotein complex. Aim 1 will determine: (a) how miR-122 base-pairs with (+)- strand HCV RNA within the MASC complex; (b) whether RNA stabilization and MASC-increased translation can be functionally uncoupled, and; (c) whether MASC function requires HCV sequences outside the response element. Aim 2 will: (a) combine RNA affinity selection and quantitative proteomics analysis to identify protein components of the MASC complex; (b) validate the presence of these proteins in the MASC complex by IP and assess their role in miR-122 function by RNA interference; (c) develop a cell-free system in which MASC function is recapitulated and the role of host proteins can be further characterized, and; (d) ascertain whether HCV RNA is stabilized by tethering of Ago or other proteins to the 5' RNA. Aim 3 will determine: (a) whether miR-122 regulates macroassembly or stability of replication vesicles by live cell imaging, (b) whether miR-122 is required for negative-strand RNA synthesis, and (c) whether miR-122 facilitates separation of duplexed strands within replicative forms of HCV RNA. Collectively, these studies will provide novel insights into functions of a miRNA that: (a) are important in the pathogenesis of human disease, (b) represent a well-validated but poorly understood therapeutic target, and (c) are unique not only among mammalian viruses but among the Metazoa. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a major cause of liver-specific morbidity and mortality worldwide, resulting in >350,000 deaths annually due to cancer and cirrhosis with especially high rates of disease among those co-infected with human immunodeficiency virus (HIV) or having a history of injection drug use. This project will investigate the novel role played by a small cellular ribonucleic acid, miR-122, in this disease. This liver-specific micro-RNA is essential for replication of HCV, and it is a well-validated but very poorly understood target for antiviral therapy. The studies proposed will provide novel insight into how it functions in HCV replication and in the pathogenesis of chronic hepatitis C, thereby enhancing the scientific background on which it can be exploited as a therapeutic target.

描述（由申请人提供）：丙型肝炎病毒（HCV）是全球肝脏特异性发病率和死亡率的主要原因，由于肝硬化和癌症的疾病率高于与人类免疫缺陷病毒（HIV）共同感染的患者，每年导致> 350,000例死亡。 MicroRNA-122（miR-122），一种丰富的肝特异性miRNA对于复制感染性HCV至关重要，因此在慢性乙型肝炎的发病机理中起着新作用。最近的黑猩猩研究已验证了miR-122的miR-122沉默作为有效的抗病毒策略，却如何促进mir-122促进HCV的复制，并尚未很好地理解。最近的工作表明，miR-122通过在（+） - 链RNA的5'末端与miRNA响应元件进行直接相互作用来促进病毒翻译，而新的初步数据也表明它也可以稳定RNA。该项目将研究以下假设：MiR-122与AGO和可能其他细胞蛋白相关，形成核糖核蛋白复合物（+） - 链HCV RNA的5'端的核糖核蛋白复合物（与MiRNA相关的稳定复合物”或MASC。 MASC复合物具有多种提出的功能：（i）它保护（+） - 细胞质区室内的链RNA免受降解，从而增加可用于翻译和组装到膜结合复制酶复合酶配合物中的RNA； （ii）它可以直接增强HCV RNA的翻译活性； （iii），它可以通过促进复制酶复合物的宏观组装和/或从头开始RNA合成来促进新的病毒RNA合成。一系列相互关联的目标将严格检验这些假设，确定MASC的RNA和蛋白质成分，并进一步表征这种独特的核糖核蛋白蛋白复合物的新功能。 AIM 1将确定：（a）MiR-122碱基对（+） - 链HCV RNA在MASC络合物中如何； （b）RNA稳定和MASC增强的翻译是否可以在功能上取消耦合，并且； （c）MASC功能是否需要响应元素之外的HCV序列。 AIM 2将：（a）结合RNA亲和力选择和定量蛋白质组学分析，以鉴定MASC复合物的蛋白质成分； （b）通过IP验证这些蛋白在MASC复合物中的存在，并通过RNA干扰评估它们在miR-122功能中的作用； （c）开发一个无细胞的系统，其中概括了MASC功能，并且可以进一步表征宿主蛋白的作用，并且； （d）确定HCV RNA是否通过将AGO或其他蛋白质固定在5'RNA中稳定。 AIM 3将确定：（a）miR-122是否通过活细胞成像调节复制囊泡的宏观组装或稳定性，（b）负链RNA合成是否需要miR-122，以及（c）miR-122是否促进了在HCV RNA复制形式内的双链链分离。总的来说，这些研究将提供有关miRNA功能的新见解，该功能：（a）在人类疾病的发病机理中很重要，（b）代表了一个有效但知之甚少的治疗靶标，并且（c）在哺乳动物病毒中不仅是独特的，而且在梅托唑中是独一无二的。 公共卫生相关性：丙型肝炎病毒（HCV）是全球肝脏特异性发病率和死亡率的主要原因，每年由于癌症和肝硬化而导致> 350,000人死亡，与人类免疫缺陷病毒（HIV）共同感染的人（尤其是疾病）尤其很高，或者具有注射药物使用史。该项目将调查小细胞核糖核酸miR-122在该疾病中起的新作用。该肝特异性微-RNA对于复制HCV至关重要，它是一个验证良好但知之甚少的抗病毒药治疗靶标。提出的研究将提供有关其在HCV复制和慢性丙型肝炎发病机理中如何发挥作用的新见解，从而增强了可以利用其作为治疗靶点的科学背景。