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(丙型肝炎病毒)是全球肝脏特有发病率和死亡率的主要原因，每年导致350,000人死于肝硬变和癌症，在那些同时感染人类免疫缺陷病毒(HIV)或有注射吸毒史的人中，发病率很高。MicroRNA-122(miR-122)是一种丰富的、肝脏特异的miRNA，对于感染性丙型肝炎的复制是必不可少的，因此在慢性丙型肝炎的发病机制中发挥了新的作用。最近的黑猩猩研究证实，沉默miR-122是一种有效的抗病毒策略，但miR-122如何促进丙型肝炎病毒复制尚不清楚。最近的工作表明，miR-122通过与(+)链RNA 5‘端的miRNA反应元件直接相互作用促进病毒翻译，而新的初步数据表明，它也起到稳定RNA的作用。该项目将调查这一假设，即miR-122与AGO和可能的其他细胞蛋白一起，在(+)链丙型肝炎病毒RNA的5‘端形成核糖核蛋白复合体(“miRNA相关稳定复合体”，或MASC)。MASC复合体有几个拟议的功能：(I)它保护细胞质中的(+)-链RNA不被降解，从而增加可用于翻译和组装成膜结合的复制酶复合体的RNA；(Ii)它可以直接增强丙型肝炎病毒RNA的翻译活性；(Iii)它可能通过促进复制酶复合体的大组装和/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复合体的新功能。目标1将决定：(A)miR-122碱基如何与MASC复合体中的(+)链丙型肝炎病毒RNA配对；(B)RNA稳定和MASC增加的翻译是否可以在功能上分离，以及(C)MASC功能是否需要反应元件外的丙型肝炎病毒序列。目的2将：(A)将RNA亲和力选择和定量蛋白质组学分析相结合，以确定MASC复合体的蛋白质组分；(B)通过IP验证这些蛋白质在MASC复合体中的存在，并通过RNA干扰评估它们在miR-122功能中的作用；(C)建立一个无细胞系统，在该系统中，MASC功能得到重现，宿主蛋白的作用可以进一步表征；以及(D)确定丙型肝炎病毒RNA是否通过将AGO或其他蛋白质拴在5‘RNA上来稳定。目标3将决定：(A)miR-122是否通过活细胞成像调节复制小泡的大组装或稳定性，(B)miR-122是否是负链RNA合成所必需的，以及(C)miR-122是否促进复制形式的丙型肝炎病毒RNA中双链的分离。总的来说，这些研究将为miRNA的功能提供新的见解：(A)在人类疾病的发病机制中很重要，(B)代表一个经过充分验证但缺乏了解的治疗靶点，以及(C)不仅在哺乳动物病毒中，而且在后生动物中都是独一无二的。 公共卫生相关性：丙型肝炎病毒(丙型肝炎病毒)是全球肝脏特有发病率和死亡率的主要原因，每年导致350,000人死于癌症和肝硬变，在合并感染人类免疫缺陷病毒(HIV)或有注射吸毒史的人中，发病率尤其高。这个项目将调查一种小细胞核糖核酸miR-122在这种疾病中所起的新作用。这种肝脏特异的微小RNA对于复制丙型肝炎病毒是必不可少的，它是一个得到很好验证但却知之甚少的抗病毒治疗靶点。提出的研究将为它如何在丙型肝炎病毒复制和慢性丙型肝炎的发病机制中发挥作用提供新的见解，从而增强它作为治疗靶点的科学背景。