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

Abstract

Approximately 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS corona virus 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.

Keywords: (+) ssRNA; RNA; RNA structure; coronavirus; coronavirus disease 2019 (COVID-19); inhibitor; mRNA pseudoknot; programmed −1 ribosomal frameshifting (−1 PRF); small molecule inhibitor; translation; virus.

Figure 1.

Structural comparison of the SARS-CoV and SARS-CoV-2 −1 PRF signals. A, cartoon depicting SARS-CoV and SARS-CoV-2 genome organization including a −1 PRF between ORF1a and ORF1b. B, pairwise analysis of the two −1 PRF signals. The attenuator elements and three-stemmed pseudoknot sequences are boxed as indicated. The U UUA AAC slippery site is underlined. C, structure of the SARS-CoV −1 PRF signal (11) is composed of the 5′ slippery site, a 6-nt spacer, and the three-stemmed pseudoknot stimulatory element. The single-base difference in SARS-CoV-2 (red) maps to the short loop linking stems 2 and 3. D, comparison of the SARS-CoV and SARS-CoV-2 −1 PRF attenuator elements. SARS-CoV-2–specific bases are indicated in red. E and F, silent coding mutations designed to disrupt the attenuators, slippery sites, and stems 1, 2, and 3 in the SARS-CoV-2 (E) and SARS-CoV (F) −1 PRF signals. gRNA, genomic RNA.

Figure 2.

Functional characterization of the SARS-CoV and SARS-CoV-2 −1 PRF signals. A and B, analyses of silent slippery site mutants. The efficiencies of −1 PRF promoted by the WT (U UUA AAC) and silent slippery site mutant (C CUC AAC) −1 PRF signals were assayed in HEK (A) and HeLA (B). ssM denotes silent slippery site mutant. C–E, analyses of the importance of the three stems in the −1 PRF stimulating RNA pseudoknot. Silent stem 1 (St-1, C), stem 2 (St-2, D), and stem 3 (St-3, E) mutants were assayed in HEK cells. F and G, analyses of the attenuator hairpins. AH denotes constructs that included attenuator hairpin sequences. AH mutant denotes mutants harboring the silent coding attenuator hairpin sequences shown in Fig. 1 (E and F). Assays were performed using Dual-Luciferase assays as previously described (15, 16). Each data point represents a single biological replicate comprised of three technical replicates. Error bars denote S.E. n.s, not significant.

Figure 3.

Small-molecule ligand MTDB inhibits −1 PRF stimulation by SARS-CoV-2 pseudoknot. −1 PRF efficiency was reduced almost 60% in the presence of 5 μm MTDB (right), compared with −1 PRF efficiency in the absence of MTDB (left).

Figure 4.

SAXS analyses. A, scattering profiles from laboratory-purified SAXS samples containing pseudoknots from SARS-CoV (blue) and SARS-CoV-2 (red). Inset, scattering profiles from inline SEC-SAXS measurements, containing purely monomeric pseudoknots. B and C, difference between the scattering profiles for SARS-CoV and SARS-CoV-2 pseudoknots obtained from lab-purified SAXS (B) and inline SEC-SAXS (C) samples. Arb., arbitrary.