Micro-RNA 122 and Chronic Hepatitis C

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碱基对如何与MASC复合体内的()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。

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.