Micro-RNA 122 and Chronic Hepatitis C

Micro-RNA 122 和慢性丙型肝炎

• 批准号: 8444519
• 负责人: Stanley M. Lemon
• 金额: $ 34.63万
• 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/8444519
• 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; Fluorescence Resonance Energy Transfer; Genetic; Genome; HIV; Hepatitis C virus; Immunoprecipitation; Infectious hepatitides; 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; injection drug use; 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.

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。