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

针对丙型肝炎病毒(丙型肝炎病毒)的新的直接作用的抗病毒药物可以治愈大多数患者的病毒。然而，这些 化合物价格昂贵，全球1.7亿感染艾滋病毒的人中的大多数人都无法获得 丙型肝炎。也没有批准的疫苗或抗病毒化合物用于黄病毒科的其他成员，如 如登革热病毒、西尼罗河病毒或寨卡病毒。因此，对病毒至关重要的细胞基因的鉴定 传播非常重要，因为这样的靶点不受容易出错的病毒RNA的调节 因此，聚合酶提供了很高的抗药性屏障。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，显示亲病毒或 抗病毒活动。众所周知，microRNA miR-122以寡聚复合体的形式附着在 病毒基因组并保护其免受宿主核酸外切酶的降解。奇怪的是，逃脱了藏匿着 在122-Anagomir治疗的患者血清中检测到病毒基因组中的单一C3U突变。这个 第一个目标的总体目标是确定C3U丙型肝炎病毒基因组与核酸或 当miR-122丰度降低时，允许病毒持续存在的蛋白质。中心假设是 C3U丙型肝炎病毒基因组中新的RNA-RNA或蛋白质RNA相互作用允许稳定和表达 在miR-122丰度降低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分析方法 检测三级RNA结构的方法将被用于研究RNA-蛋白质和RNA-RNA 培养肝细胞中野生型和突变型病毒RNA的相互作用。病毒生命周期中的以下步骤 受这种核酸-蛋白质相互作用调节的蛋白质将被识别。第二个目标的总体目标是 基于宿主细胞衍生的环状RNA(CRNAs)在丙型肝炎病毒感染期间发生改变的发现 感染。将探索CRNAs发挥其促病毒和抗病毒功能的潜在机制。 首先，cRNA介导的蛋白质和microRNAs对丙型肝炎病毒rna扩增的影响将是 检查过了。因为CRNAs中特定腺苷的甲基化已被证明可以诱导翻译 在CRNAs中启动，将确定CRNAs中的甲基化状态。甲基化的CRNAs的特性 合成小肽将使用遗传和蛋白质组学方法进行检测。总体而言，应用程序 详细介绍了研究非编码RNA在病毒-宿主相互作用中的作用的创新范式转换概念， 使用新的检测方法。这一提议的基本原理是非编码RNA，如 MicroRNAs和CRNAs影响丙型肝炎病毒的致病机制。已经证明，在丙型肝炎病毒中可以靶向microRNAs 患者，导致病毒RNA丰度的损失。因此，靶向肝脏中的亲病毒CRNAs提供了一种新的 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.