Tavares Rafael de Cesaris Araujo, Mahadeshwar Gandhar, Wan Han, Huston Nicholas C, Pyle Anna Marie
Department of Chemistry, Yale University, New Haven, CT 06511, USA.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA.
J Virol. 2021 Mar 1;95(5). doi: 10.1128/JVI.02190-20. Epub 2020 Dec 2.
SARS-CoV-2 is the causative viral agent of COVID-19, the disease at the center of the current global pandemic. While knowledge of highly structured regions is integral for mechanistic insights into the viral infection cycle, very little is known about the location and folding stability of functional elements within the massive, ∼30kb SARS-CoV-2 RNA genome. In this study, we analyze the folding stability of this RNA genome relative to the structural landscape of other well-known viral RNAs. We present an in-silico pipeline to predict regions of high base pair content across long genomes and to pinpoint hotspots of well-defined RNA structures, a method that allows for direct comparisons of RNA structural complexity within the several domains in SARS-CoV-2 genome. We report that the SARS-CoV-2 genomic propensity for stable RNA folding is exceptional among RNA viruses, superseding even that of HCV, one of the most structured viral RNAs in nature. Furthermore, our analysis suggests varying levels of RNA structure across genomic functional regions, with accessory and structural ORFs containing the highest structural density in the viral genome. Finally, we take a step further to examine how individual RNA structures formed by these ORFs are affected by the differences in genomic and subgenomic contexts, which given the technical difficulty of experimentally separating cellular mixtures of sgRNA from gRNA, is a unique advantage of our in-silico pipeline. The resulting findings provide a useful roadmap for planning focused empirical studies of SARS-CoV-2 RNA biology, and a preliminary guide for exploring potential SARS-CoV-2 RNA drug targets. The RNA genome of SARS-CoV-2 is among the largest and most complex viral genomes, and yet its RNA structural features remain relatively unexplored. Since RNA elements guide function in most RNA viruses, and they represent potential drug targets, it is essential to chart the architectural features of SARS-CoV-2 and pinpoint regions that merit focused study. Here we show that RNA folding stability of SARS-CoV-2 genome is exceptional among viral genomes and we develop a method to directly compare levels of predicted secondary structure across SARS-CoV-2 domains. Remarkably, we find that coding regions display the highest structural propensity in the genome, forming motifs that differ between the genomic and subgenomic contexts. Our approach provides an attractive strategy to rapidly screen for candidate structured regions based on base pairing potential and provides a readily interpretable roadmap to guide functional studies of RNA viruses and other pharmacologically relevant RNA transcripts.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）是冠状病毒病（COVID-19）的致病病毒病原体，而COVID-19是当前全球大流行的核心疾病。虽然对于病毒感染周期的机制性洞察而言，了解高度结构化区域不可或缺，但对于庞大的、约30kb的SARS-CoV-2 RNA基因组中功能元件的位置和折叠稳定性却知之甚少。在本研究中，我们分析了该RNA基因组相对于其他知名病毒RNA结构景观的折叠稳定性。我们提出了一种计算机模拟流程，用于预测长基因组中高碱基对含量区域，并精确确定明确RNA结构的热点，该方法能够直接比较SARS-CoV-2基因组中几个结构域内的RNA结构复杂性。我们报告称，SARS-CoV-2基因组形成稳定RNA折叠的倾向在RNA病毒中是异常的，甚至超过了丙型肝炎病毒（HCV），HCV是自然界中结构最复杂的病毒RNA之一。此外，我们的分析表明，整个基因组功能区域的RNA结构水平各不相同，辅助和结构开放阅读框在病毒基因组中具有最高的结构密度。最后，鉴于从实验上分离亚基因组RNA（sgRNA）与基因组RNA（gRNA）的细胞混合物存在技术困难，我们进一步研究了这些开放阅读框形成的单个RNA结构如何受到基因组和亚基因组环境差异的影响，这是我们计算机模拟流程的独特优势。所得结果为规划针对SARS-CoV-2 RNA生物学的重点实证研究提供了有用的路线图，并为探索潜在的SARS-CoV-2 RNA药物靶点提供了初步指导。SARS-CoV-2的RNA基因组是最大且最复杂的病毒基因组之一，但其RNA结构特征仍相对未被探索。由于RNA元件在大多数RNA病毒中指导功能，且它们代表潜在的药物靶点，因此描绘SARS-CoV-2的结构特征并确定值得重点研究的区域至关重要。在此我们表明SARS-CoV-2基因组的RNA折叠稳定性在病毒基因组中是异常的，并且我们开发了一种方法来直接比较SARS-CoV-2各结构域预测二级结构的水平。值得注意的是，我们发现编码区域在基因组中表现出最高的结构倾向，形成了在基因组和亚基因组环境中不同的基序。我们的方法提供了一种有吸引力的策略，可基于碱基配对潜力快速筛选候选结构化区域，并提供了一个易于解读的路线图，以指导RNA病毒和其他药理学相关RNA转录本的功能研究。
