Micro-RNA 122 and Chronic Hepatitis C

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，每年因肝硬化和癌症导致> 350，000例死亡，在合并感染人类免疫缺陷病毒（HIV）或有注射吸毒史的人群中发病率较高。microRNA-122（miR-122）是一种丰富的肝脏特异性miRNA，是感染性HCV复制所必需的，因此在慢性丙型肝炎的发病机制中起着新的作用。最近的黑猩猩研究已经验证了miR-122沉默作为一种有效的抗病毒策略，但miR-122如何促进HCV复制还不清楚。最近的研究表明，miR-122通过与（+）-链RNA的5'端的miRNA反应元件直接相互作用来促进病毒翻译，而新的初步数据表明，它还起到稳定RNA的作用。该项目将研究miR-122与Ago和可能的其他细胞蛋白结合在（+）-链HCV RNA的5'端形成核糖核蛋白复合物（“miRNA相关稳定复合物”，或MASC）的假设。MASC复合物具有几种功能：（i）它保护细胞质区室中的（+）-链RNA免于降解，从而增加可用于翻译和组装成膜结合复制酶复合物的RNA;（ii）它可以直接增强HCV RNA的翻译活性;和（iii）它可以通过促进复制酶复合物的大组装和/或RNA合成的从头起始来促进新的病毒RNA合成。一系列相互关联的目标将严格测试这些假设，确定MASC的RNA和蛋白质组分，并进一步表征这种独特的核糖核蛋白复合物的新功能。目标1将确定：（a）miR-122如何与MASC复合物内的（+）-链HCV RNA碱基配对;（B）RNA稳定化和MASC增加的翻译是否可以在功能上解偶联;和（c）MASC功能是否需要应答元件外的HCV序列。目标2将：（a）将联合收割机RNA亲和力选择和定量蛋白质组学分析相结合，以鉴定MASC复合物的蛋白质组分;（B）通过IP验证这些蛋白质在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）开发一种无细胞系统，其中MASC功能被概括，宿主蛋白的作用可以进一步表征，和;（d）确定HCV RNA是否通过Ago或其它蛋白质与5' RNA的连接而稳定。目标3将确定：（a）通过活细胞成像，miR-122是否调节复制囊泡的宏组装或稳定性，（B）miR-122是否是负链RNA合成所需的，和（c）miR-122是否促进HCV RNA复制形式内双链的分离。总的来说，这些研究将提供对miRNA功能的新见解：（a）在人类疾病的发病机制中是重要的，（B）代表了一个经过充分验证但知之甚少的治疗靶点，（c）不仅在哺乳动物病毒中而且在后生动物中是独特的。

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.