Roles for microRNA-122 and circular RNAs in flavivirus RNA amplification

新的直接作用的抗丙型肝炎病毒（HCV）的抗病毒药物治愈了大多数患者的病毒。但这些 化合物是昂贵的，并且对于全世界1.7亿感染者中的大多数人来说是不可用的。 丙肝病毒。对于黄病毒科的其他成员，也没有批准的疫苗或抗病毒化合物，如 登革热病毒西尼罗河病毒或寨卡病毒。因此，鉴定病毒所必需的细胞基因， 传播是非常重要的，因为这些目标不受易错病毒RNA的调节 聚合酶，并因此提供耐药性的高屏障。这个应用程序的长期目标是探索 某些非编码RNA，如microRNA和环状RNA，显示前病毒或 抗病毒活性。已知微小RNA miR-122作为寡聚复合物连接到微RNA的5'末端。 病毒基因组，并保护它免受宿主核酸外切酶的降解。奇怪的是，拥有一个 在接受122-氯米松治疗的患者的血清中检测到病毒基因组中的单个C3 U突变。的 第一个目的的总体目标是鉴定C3 U HCV基因组与核酸之间的相互作用，或 当miR-122丰度降低时，允许病毒持续存在的蛋白质。核心假设是， C3 U HCV基因组中新的RNA-RNA或蛋白质RNA相互作用允许C3 U HCV基因组的稳定和表达， 在降低的miR-122丰度期间细胞中的病毒基因组。新的基于细胞的蛋白质生物素化测定 而检测RNA三级结构的方法将被用于研究RNA-蛋白质和RNA-RNA 在培养的肝细胞中野生型和突变型病毒RNA的相互作用。病毒生命周期中的步骤 将鉴定由这种核酸-蛋白质相互作用调节的蛋白质。第二个目标的总体目标是 基于发现宿主细胞来源的环状RNA（cRNA）在HCV感染期间改变， 感染cRNA发挥其促病毒和抗病毒功能的潜在机制将被探索。 首先，将研究cRNA介导的蛋白质和microRNA的隔离对HCV RNA扩增的影响。 考察因为cRNA中特定腺苷的甲基化已被证明可以诱导翻译 在cRNA中的甲基化起始之后，将确定cRNA中的甲基化状态。甲基化cRNA的性质， 合成小肽将使用遗传学和蛋白质组学方法进行检查。总的来说，应用 详细介绍了创新的范式转变概念，以研究非编码RNA在病毒-宿主相互作用中的作用， 使用新颖的检测方法。这一提议的基本原理是，非编码RNA，如 microRNA和cRNA影响HCV发病机制。已经表明，microRNA可以在HCV中靶向 患者，导致病毒RNA丰度的损失。因此，靶向肝脏中的前病毒cRNA提供了一种新的治疗方法。 抗病毒策略

New direct-acting antivirals against hepatitis C virus (HCV) cure the virus in most patients. However, these compounds are expensive and not available to most of the 170 million people that are worldwide infected with HCV. There are also no approved vaccines or anti-viral compounds for other members of the flaviviridae, such as Dengue virus, West Nile virus or Zika virus. Thus, identification of cellular genes that are essential for virus propagation is highly significant, because such targets are not regulated by the error-prone viral RNA polymerase and, therefore, offer a high barrier to resistance. The long-term goal of this application is to explore the mechanism by which certain noncoding RNAs, such as microRNAs and circular RNAs, display pro-viral or anti-viral activities. It is known that microRNA miR-122 attaches as an oligomeric complex to the 5’ end of the viral genome and protects it from degradation by host exonucleases. Curiously, escape mutants that harbor a single C3U mutation in the viral genome were detected in the serum of 122-antagomir treated patients. The overall objective of the first aim to is identify interactions of between C3U HCV genome with nucleic acids or proteins that allow the virus to persist when miR-122 abundance is reduced. The central hypothesis is that novel RNA-RNA or protein RNA interactions in the C3U HCV genome allow stabilization and expression of the viral genome in the cells during reduced miR-122 abundances. Novel cell-based protein biotinylation assays and approaches that detect tertiary RNA structures will be used to study RNA-protein and RNA-RNA interactions in wildtype and mutant viral RNAs in cultured liver cells. Steps in the viral life cycle that are modulated by such nucleic acid-protein interactions will be identified. The overall objective of the second aim is based upon the finding that the host cell-derived circular RNA (cRNAs) landscape is altered during HCV infection. The potential mechanisms by which cRNAs exert their pro- and antiviral functions will be explored. First, effects of cRNA-mediated sequestration of proteins and microRNAs on HCV RNA amplification will be examined. Because methylation of specific adenosines in cRNAs has been shown to induce translation initiation in cRNAs, the methylation status in cRNAs will be determined. The properties of methylated cRNAs to synthesize small peptides will be examined using genetic and proteomic approaches. Overall, the application details innovative paradigm-shifting concepts to study roles for noncoding RNAs in virus-host interactions, using novel detection methodologies. The rationale for this proposal is that noncoding RNAs, such as microRNAs and cRNAs, affect HCV pathogenesis. It has been shown that microRNAs can be targeted in HCV patients, resulting in loss of viral RNA abundance. Thus, targeting pro-viral cRNAs in the liver offers a novel antiviral strategy.