Micro-RNA 122 and Chronic Hepatitis C

 描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)，一种(+)链RNA病毒，持续感染全球约2亿人，尽管抗病毒治疗取得了进展，但仍是美国肝硬变和肝癌的主要原因。在本申请中，我们 建议继续进行一系列富有成效的研究，旨在了解丙型肝炎病毒复制独特依赖于进化保守的、肝脏特异的宿主microRNA miR-122-5p的分子机制(S)。我们已发表的研究表明，miR-122与9.7kb(+)正义RNA基因组5‘端附近的两个保守位点(S1和S2)的结合以两种不同的方式促进丙型肝炎病毒的复制：(1)通过与S1和S2结合，miR-122保护丙型肝炎病毒RNA免受XRN1(细胞质宿主5’外切核糖核酸酶)介导的5‘外切核变性；(2)miR-122通过重新平衡参与模板RNA合成的(+)-RNA分子的比例而直接刺激丙型肝炎病毒RNA的合成。这两种功能都需要AGO2，我们的数据显示，AGO2是由miR-122招募到基因组中的。我们现在在新的初步数据中表明，miR-122还通过竞争与多聚(RC)结合蛋白2(PCBP2)以及可能的其他宿主RNA结合蛋白来调节丙型肝炎病毒基因组的循环。我们提出了三个具体的目标来研究这一总体假设，即由miR-122和AGO2在丙型肝炎病毒基因组5‘端形成的独特的核糖核蛋白复合体在病毒生命周期的不同阶段关键地调节其参与。在目标1中，我们将评估miR-122在调节单链特异性丙型肝炎病毒RNA合成和新合成的(+)-RNA包装中的作用。目的2将研究miR-122与宿主RNA结合蛋白(包括PCBP2和通过定量蛋白质组学鉴定的新发现的候选宿主因子)之间的竞争如何调节病毒复制。目的3将表征miR-122在通过蛋白质-蛋白质相互作用调节丙型肝炎病毒(+)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。虽然我们的努力主要集中在丙型肝炎病毒上，但这些研究与其他致病(+)链RNA病毒的复制机制具有广泛的相关性。

 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.