Novel RNA Structures in Coronavirus Replication

描述(申请人提供)：该项目的长期目标是详细了解这些关键的顺式作用5‘和3’UTRs的高分辨率RNA结构，以及指导SARS冠状病毒(SARS-CoV)和密切相关的第2组冠状病毒-小鼠肝炎病毒(MHV)复制和繁殖的蛋白质-RNA相互作用。这些信息对于最终开发可用于消除冠状病毒复制的特定抗病毒药物是不可或缺的。为此，我们开发了一个基于协变的冠状病毒5‘UTR二级结构模型，该模型包含三个保守的茎环结构，SL1，SL2和SL4。这一模型为设计实验和开发假说提供了基础，这些假说将导致对RNA结构和RNA-蛋白质相互作用如何调控冠状病毒复制的新的分子水平的洞察。这个应用程序主要关注我们模型的三个方面。在第一个目标中，我们将调查我们的假设，即SL1必须是一个动态结构，以允许与其他RNA序列和/或复制辅助蛋白相互作用，最终在基因组环化中介导5‘和3’UTRs之间的相互作用。将利用生化、生物物理(核磁共振、热力学研究)和反向遗传方法来确定SL1的稳定性，以及在病毒复制中对动态SL1的需求。将进行生化研究，以确定与SL1结合的蛋白质及其在冠状病毒基因组循环中的作用。在第二个目标中，我们将研究SL2，我们的初步研究表明，它采用了一种不寻常的U型转弯结构。我们建议使用核磁共振方法以高分辨率求解SL2的溶液结构，并进行一系列反向遗传学研究，以测试我们的结构模型在MHV基因组背景下的预测，并确定它们对病毒复制的影响。在第三个目的中，我们将采用生物物理和反向遗传实验相结合的方法来研究冠状病毒核衣壳蛋白与5‘端转录调控序列(TRS-L)的相互作用。我们将通过核磁共振波谱和生物物理方法确定核衣壳蛋白：TrS-L RNA复合物的溶液结构。受核衣壳蛋白：TRS-L结构的影响，我们将进行一系列反向遗传学研究，以确定TRS：核衣壳相互作用是否在RNA复制、亚基因组RNA合成和/或翻译中发挥作用。这些研究将极大地促进我们对冠状病毒关键顺式作用序列和相互作用蛋白的详细结构和功能的理解，并为冠状病毒的复制提供机械性的见解。 冠状病毒是人类和兽医的重要病原体，其中SARS冠状病毒是最严重的人类病原体。尽管最初的SARS疫情已得到控制，但这种病毒有可能从其人畜共患病源头重新出现。本申请中提出的研究将促进我们对冠状病毒复制的详细了解，并通过这样做为未来针对这组病毒开发更多的抗病毒药物奠定基础。

DESCRIPTION (provided by applicant): The long-term goal of this project is to obtain a detailed understanding of the high resolution RNA structures of these critical cis-acting 5' and 3' UTRs and of protein-RNA interactions that direct the replication and propagation of SARS-coronavirus (SARS-CoV) and the closely related group 2 coronavirus, mouse hepatitis virus (MHV). Such information is integral to the eventual development of specific antiviral agents that could be used to abrogate coronavirus replication. Towards that end we have developed a covariation-based model of the secondary structure of the coronavirus 5' UTR that contains three conserved stem-loop structures, SL1, SL2, and SL4. This model has provided the basis for designing experiments and developing hypotheses that will lead to new molecular-level insights into how RNA structure and RNA-protein interactions regulate coronavirus replication. This application focuses on three aspects of our model. In the first aim we will investigate our hypothesis that SL1 must be a dynamic structure to allow interactions with other RNA sequences and/or replication accessory proteins that ultimately mediate an interaction between the 5' and 3' UTRs in genome circularization. Biochemical, biophysical (NMR, thermodynamic studies), and reverse genetic approaches will be utilized to determine the stability of SL1, and the requirement for a dynamic SL1 in viral replication. Biochemical studies will be performed to identify proteins that bind to SL1 and their role in circularization of the coronavirus genome. In the second aim we will investigate SL2, a stem-loop that our preliminary studies have indicated adopts an unusual U-turn like structure. We propose to solve the solution structure of SL2 at high resolution using NMR methods, and perform a series of reverse genetic studies to test predictions of our structural model in the context of the MHV genome and determine their effects on viral replication. In the third aim we will employ a combination of biophysical and reverse genetic experiments to investigate the interaction of the coronavirus nucleocapsid protein with the transcriptional regulatory sequences in the 5' leader (TRS-L) RNA. We will determine the solution structure of nucleocapsid protein:TRS-L RNA complexes by NMR spectroscopy and biophysical methods. Informed by the nucleocapsid protein:TRS-L RNA structure, we will perform a series of reverse genetic studies to determine if the TRS:nucleocapsid interaction plays a role in RNA replication, subgenomic RNA synthesis and/or translation. The proposed studies will significantly advance our understanding of the detailed structure and function of critical coronavirus cis- acting sequences and interacting proteins, and provide mechanistic insights into coronavirus replication. Coronaviruses are important human and veterinary pathogens, with the SARS-coronavirus being the most serious human pathogen. Although the original outbreak of SARS was brought under control, this virus is a threat to re-emerge from its zoonotic source. The research proposed in this application will advance our detailed knowledge of coronavirus replication, and by doing so lay the basis for future development of additional anti-virals directed against this group of viruses.