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

Abstract

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. Importance 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.

Supramolecular cylinders target bulge structures in the 5’ UTR of the RNA genome of SARS-CoV-2 and inhibit viral replication

“…The additional stem-loop (SL4) predicted by RNAfold but not Mxfold is acylated (region K) which suggests that if such a stem loop forms it may be transient. Recent studies of the whole RNA viral genome in cellulo by Miska [43] (COMRADES assay) and Pyle [44,45] (long amplicons with SHAPE-MaP) show dynamic folding and interaction between the 5' UTR and 3' UTR, but that these key stem-loop structures (SL1,2,3,5; depicted in Fig2) are retained, affording further support and confidence that our in vitro findings are physiologically relevant.…”

Understanding the outcomes of COVID-19 – does the current model of an acute respiratory infection really fit?

“…One prominent feature of SARS-CoV-2 and of all other coronaviruses infecting mammals and birds is the presence of large scale, pervasive RNA secondary structure throughout the genome [28]. Although a subset of structures relate to transcriptional- or replication-associated functions, such as the frameshift site in ORF1a/ORF1b, the wider distribution of RNA folding throughout the genome [28, 72] may correspond to the previously described genome-scale ordered RNA structure (GORS) in positive-stranded mammalian and avian RNA viruses. Possession of GORS is strongly associated with host persistence, although the mechanism(s) behind this association are largely uncharacterized [73, 74].…”

Structure and regulation of coronavirus genomes: state-of-the-art and novel insights from SARS-CoV-2 studies

“…Besides the well-characterized structures within the 5 0 and 3 0 UTR, and the FSE, the SARS-CoV-2 genome is predicted to have an exceptionally high propensity to form stable RNA structures, significantly higher compared with that of other RNA viruses, including Hepatitis C virus (HCV), one of the most highly structured viral RNAs to date [72]. Indeed, recent genome-scale structure analyses of the SARS-CoV-2 genome, both in vitro and in living infected host cells, have led to the identification of a plethora of novel highly stable RNA structure elements, spread along the whole genome.…”