Micro-RNA 122 and Chronic Hepatitis C

 描述（由申请人提供）：丙型肝炎病毒（HCV）是一种（+）链RNA病毒，持续感染全世界约2亿人，尽管抗病毒治疗取得了进展，但仍是美国肝硬化和肝癌的主要原因。 建议继续进行一系列富有成效的研究，旨在了解HCV复制对进化上保守的肝脏特异性宿主microRNA miR-122- 5 p的独特依赖性的分子机制。我们发表的研究表明，miR-122与两个保守位点的结合（S1和S2）在9.7kb（+）-正义RNA基因组的5'末端附近以两种不同的方式促进HCV复制：（1）通过结合S1和S2，miR-122保护HCV RNA免受XRN 1（一种细胞质宿主5'核糖核酸外切酶）介导的5'核酸外切降解，和（2）miR-122通过重新平衡参与模板RNA合成与指导核糖体翻译的（+）-RNA分子的比例直接刺激HCV RNA合成。这两种功能都需要AGO 2，我们的数据显示AGO 2被miR-122募集到基因组中。我们现在在新的初步数据中显示，miR-122还通过与多聚（rC）结合蛋白2（PCBP 2）和可能的其他宿主RNA结合蛋白竞争结合来调节HCV基因组的环化。我们提出了三个具体的目标来研究总体假设，即由miR-122和AGO 2在HCV基因组的5'端形成的独特的核糖核蛋白复合物在病毒生命周期的不同阶段严格地调节其参与。在目标1中，我们将评估miR-122在调节链特异性HCV RNA合成和包装新合成的（+）-RNA中的作用。目的2将研究miR-122和宿主RNA结合蛋白（包括PCBP 2和通过定量蛋白质组学鉴定的新识别的候选宿主因子）之间的竞争如何调节病毒复制。目的3将描述miR-122在调节蛋白质-蛋白质相互作用介导的HCV（+）链基因组环化中的作用，以及这与基因组稳定性和病毒RNA合成的关系。虽然我们的努力集中在丙型肝炎病毒具体，这些研究有广泛的相关性，其他致病性（+）链RNA病毒的复制机制。

 DESCRIPTION (provided by applicant): Hepatitis C virus (HCV), a (+)-strand RNA virus, persistently infects ~200 million persons worldwide and, despite advances in antiviral therapy, remains the leading cause of hepatic cirrhosis and liver cancer in the U.S. In this application, we propose a continuation of a productive line of research aimed at understanding the molecular mechanism(s) underlying the unique dependence of HCV replication on the evolutionarily conserved, liver-specific host microRNA, miR-122-5p. Our published research shows that the binding of miR-122 to two conserved sites (S1 and S2) near the 5' end of the 9.7 kb (+)-sense RNA genome promotes HCV replication in two distinct ways: (1) by binding S1 and S2, miR-122 protects HCV RNA from 5' exonucleolytic decay mediated by XRN1, a cytoplasmic host 5' exoribonuclease, and (2) miR-122 directly stimulates HCV RNA synthesis by rebalancing the proportion of (+)-RNA molecules engaged in templating RNA synthesis vs. directing translation on ribosomes. Both functions require AGO2 which our data show is recruited to the genome by miR-122. We now show in new preliminary data that miR-122 also regulates circularization of the HCV genome through competition for binding with poly-(rC) binding protein 2 (PCBP2) and possibly other host RNA-binding proteins. We propose three specific aims to investigate the overarching hypothesis that a unique ribonucleoprotein complex formed by miR-122 and AGO2 at the 5' end of the HCV genome critically regulates its engagement in different stages of the viral lifecycle. In Aim 1, we will assess the role of miR-122 in regulating strand-specific HCV RNA synthesis and packaging of newly synthesized (+)-RNA. Aim 2 will study how viral replication is regulated by competition between miR-122 and host RNA-binding proteins, including PCBP2 and newly recognized candidate host factors identified through quantitative proteomics. Aim 3 will characterize the role of miR-122 in modulating circularization of the HCV (+)-strand genome mediated by protein-protein interactions, and how this relates to genome stability and viral RNA synthesis. While our efforts focus on HCV specifically, these studies have broad relevance to mechanisms of replication of other pathogenic (+)-strand RNA viruses.