Conserved RNA Secondary Structures in three Betacoronaviruses: MHV, BCoV, and MERS-CoV

 描述(申请人提供)：贝塔冠状病毒属包括两种病毒，SARS-CoV和MERS-CoV，它们分别于2002年和2012年从动物宿主中出现，导致人类严重肺部疾病。在这个应用中，我们建议研究MERS-CoV和两个相关的冠状病毒，小鼠肝炎病毒(MHV)和牛冠状病毒(BCOV)的RNA二级结构，目的是鉴定和功能表征这些病毒基因组中新的保守的RNA二级结构。在目标一中，我们将使用最先进的、高通量的方法SHAPE-MAP来生成生化数据，该数据允许创建大型RNA(如冠状病毒基因组)的高精度RNA二级结构模型。通过比较MHV、BCOV和MERS-CoV基因组的RNA二级结构，我们将寻找以前未发现的具有系统发育保守的RNA二级结构，这些结构是在病毒复制中发挥功能的强有力的候选者。在目标2中，我们将使用反向遗传方法来表征在目标1中确定的保守二级结构的MHV和MERS冠状病毒复制中的功能作用。

 DESCRIPTION (provided by applicant): The Betacoronavirus genus includes two viruses, SARS-CoV and MERS-CoV that emerged from animal reservoirs to produce severe pulmonary disease in man in 2002, and 2012, respectively. In this application we propose to study the RNA secondary structure of MERS-CoV and two related coronaviruses, mouse hepatitis virus (MHV) and bovine coronavirus (BCoV) with the goal of identifying and functionally characterizing novel conserved RNA secondary structures in the genomes of these viruses. In aim one we will employ a state-of- the-art, high throughput approach, SHAPE-MaP, to generate biochemical data that allows the creation of highly accurate models of RNA secondary structures of large RNAs, such as coronavirus genomes. Through comparison of the RNA secondary structures of the genomes of MHV, BCoV, and MERS-CoV we will seek previously undiscovered phylogenetically conserved RNA secondary structures that are strong candidates to play a functional role in viral replication. In aim 2 we will employ reverse genetic approaches to characterize the functional role in MHV and MERS-CoV replication of conserved secondary structures identified in aim 1.