Genome-wide mapping of SARS-CoV-2 RNA structures identifies therapeutically-relevant elements

Abstract

SARS-CoV-2 is a betacoronavirus with a linear single-stranded, positive-sense RNA genome, whose outbreak caused the ongoing COVID-19 pandemic. The ability of coronaviruses to rapidly evolve, adapt, and cross species barriers makes the development of effective and durable therapeutic strategies a challenging and urgent need. As for other RNA viruses, genomic RNA structures are expected to play crucial roles in several steps of the coronavirus replication cycle. Despite this, only a handful of functionally-conserved coronavirus structural RNA elements have been identified to date. Here, we performed RNA structure probing to obtain single-base resolution secondary structure maps of the full SARS-CoV-2 coronavirus genome both in vitro and in living infected cells. Probing data recapitulate the previously described coronavirus RNA elements (5′ UTR and s2m), and reveal new structures. Of these, ∼10.2% show significant covariation among SARS-CoV-2 and other coronaviruses, hinting at their functionally-conserved role. Secondary structure-restrained 3D modeling of these segments further allowed for the identification of putative druggable pockets. In addition, we identify a set of single-stranded segments in vivo, showing high sequence conservation, suitable for the development of antisense oligonucleotide therapeutics. Collectively, our work lays the foundation for the development of innovative RNA-targeted therapeutic strategies to fight SARS-related infections.

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

“…The alternative conformation of stem 1 is boxed. On the right, the same alternative conformation of stem 1 as confirmed by independent in vitro and in vivo DMS and SHAPE analyses, with reactivities superimposed from Manfredonia et al [39]. The slippery site is boxed in blue.…”

“…A highly conserved structure element, stemming from the multi-branched loop of the HVR, is the stem-loop II-like motif (s2m). This stem-loop shows extreme sequence and structure similarity to the second stem-loop from the 3 0 UTR of astroviruses and equine rhinovirus [70], and its existence in SARS-CoV-2 is supported under both in vitro and in vivo conditions by multiple studies [33,[35][36][37][38][39]. X-ray crystallography of the s2m revealed that the tertiary folding of the apical pentanucleotide GAGUA loop resembles that of a typical GNRA tetraloop, with the U bulging out of the stack [71].…”

“…A first consensus model of the 5 0 UTR was originally proposed by comparative analysis of nine CoV from the three major CoV groups (alpha, beta, and gamma-CoV), and identified the three major conserved stem-loops SL1, SL2, and SL4 [32]. All three SLs have been consistently predicted and experimentally verified in SARS-CoV-2 [33][34][35][36][37][38][39]. Besides these, up to eight SLs can be present in CoV 5 0 UTRs, with some degree of variation across different genera ( Figure 1).…”

“…Complete deletion of the uORF start codon, however, resulted in defective viruses, and was spontaneously rescued by the appearance of a new uORF, suggesting that this uORF is involved in regulating the translation of ORF1ab. In MHV, the complete disruption of the SL4 structure, as Secondary structure of the SARS-CoV-2 5 0 UTR, with superimposed in vivo SHAPE reactivities from Manfredonia et al [39]. Highly (red) and moderately (yellow) reactive residues from in vitro SHAPE (circles; Manfredonia et al [39]), in vitro DMS (triangles; Manfredonia et al [39]), in vivo SHAPE (squares; Huston et al [36]) and in vivo DMS ( pentagons; Lan et al 2020) experiments are also indicated [36,37,39].…”

“…This preference for locally stable structures over long-range interactions might be involved in safeguarding the stability of SARS-CoV-2 genome and ensuring translation fidelity. On the top, the structures of conserved secondary structure elements, supported by significant covariation, as determined by SHAPE analyses of the SARS-CoV-2 genome, with superimposed reactivities from Manfredonia et al [39] (1, 2, 6, 7, 9, 10, 17) and Huston et al [36] (3,4,5). In addition, the structures of seven stem-loops (8,11,12,13,14,15,16) proposed to enclose the TRS-Bs (boxed in green), with superimposed reactivities from Lan et al [37], are shown.…”