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

Figures

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  Figure 1.. Structural comparison of the SARS-CoV and SARS-CoV-2 −1 PRF signals. A. Cartoon depicting SARS-CoV and SARS-CoV2 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 and SARS-CoV −1 PRF signals respectively.

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  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 wild-type (U UUA AAC) and silent slippery site mutant (C CUC AAC) −1 PRF signals were assayed in HEK (panel A) and HeLA (panel 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 Figs. 1E, 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.M.

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  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 to −1 PRF efficiency in the absence of MTDB (left).

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  Figure 4.. SAXS analyses. (A) Scattering profiles from lab 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, C) Difference between the scattering profiles for SARS-CoV and SARS-CoV-2 pseudoknots obtained from lab-purified (B) and inline-SEC (C) SAXS samples.