Structural and functional conservation of the programmed −1 ribosomal frameshift signal of SARS coronavirus 2 (SARS-CoV-2)

Abstract

About 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS coronavirus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule–based interventions. All coronaviruses utilize a molecular mechanism called programmed −1 ribosomal frameshift (−1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high −1 PRF rates and that it also harbors a −1 PRF attenuation element. Altering −1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar −1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate −1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit −1 PRF was similarly effective against −1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.

Comprehensive in-vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms

“…However, our data indicate that the region corresponding to SL2 is conformationally flexible, adopting an SL2 stem with only a 20% probability. Consistent with our reported distribution of structural isoforms, Kelly et al use a reporter assay to suggest that the frequency of successful frameshifting in SARS-CoV-2 is about 20% (Kelly et al, 2020), indicating that the observed conformational variability of SL2 may be functional. Indeed, SL2 might function like a switch: When SL2 is formed (~20% of the time), frameshifting occurs.…”

“…The core of the SARS-CoV PRF, which shares an almost identical sequence with SARS-CoV-2, is predicted to form a three-stem pseudoknot comprised of SLI, SL2, and a pseudoknot helix, with an additional upstream attenuator stem that is poorly conserved in SARS-CoV-2 (Kelly et al, 2020). Our SHAPE reactivity and structure prediction are consistent with the existence of an attenuator stem, SL1, and the pseudoknot.…”

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

“…Our cryo-EM-guided data and modeling, along with recent results from several groups, suggest explanations for why FSE targeting efforts by us and others have so far yielded only modest inhibition of frameshifting and viral replication [4][5][6]34 . There is accumulating evidence that, in the full SARS-CoV-2 genome context, the FSE forms alternative structures involving genomic segments upstream of the element (Extended Data Fig.…”

“…Incomplete inactivation of coronavirus frameshifting and replication in these and prior studies motivated structural analysis to better understand the FSE. Despite prior expectations of structural heterogeneity and propensity for multimerization 4,45,52 , and despite having a size slightly under that of the previous smallest macromolecule imaged by cryo-EM ( We note two caveats regarding our cryo-EM analysis. First, the 6.9 Å resolution of the cryo-EM map is not sufficient to directly resolve base interactions, much less atom positions.…”

The short- and long-range RNA-RNA Interactome of SARS-CoV-2

“…On the full-length gRNA itself, two partially overlapping open reading frames (ORF1a and ORF1b) are translated from the same start codon at the 5′ end, resulting in the polyproteins pp1a and pp1ab. Translation of the longer product pp1ab is made possible by a hairpin-type pseudoknot RNA s tructure known as the frameshifting element (FSE) which regulates a programmed -1 ribosomal frameshifting that overrides with about 50% efficiency the stop codon of ORF1a (Kelly et al, 2020;Namy et al, 2006) . Previous studies applied RNA structure probing techniques using s elective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) and DMS, as well as nuclear magnetic resonance (NMR) to effectively identify conserved cis-acting RNA structures regulating the life cycle of coronaviruses.…”