The Global and Local Distribution of RNA Structure throughout the SARS-CoV-2 Genome

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative 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 (∼30-kb) SARS-CoV-2 RNA genome. In this study, we analyzed 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 hot spots 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 hepatitis C virus (HCV), one of the most structured viral RNAs in nature. Furthermore, our analysis suggests various levels of RNA structure across genomic functional regions, with accessory and structural open reading frames (ORFs) containing the highest structural density in the viral genome. Finally, we took 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 subgenomic RNA (sgRNA) from genomic RNA (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. IMPORTANCE The RNA genome of SARS-CoV-2 is among the largest and most complex viral genomes, 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. In this study, we found that RNA folding stability of SARS-CoV-2 genome is exceptional among viral genomes and we developed a method to directly compare levels of predicted secondary structure across SARS-CoV-2 domains. Remarkably, we found 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.