Restriction of SARS-CoV-2 Replication by Targeting Programmed −1 Ribosomal Frameshifting In Vitro

Figures

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  Figure 1. A high-throughput screen identifies SARS-CoV-2 PRF modulators. (A) Schematic illustration of the SARS-CoV-2 genome architecture, with the FSE indicated. (B) Schematic illustration of the dual luciferase-based −1 PRF reporter design. Watson-Crick base pairs are indicated by filled circles. Each of the three stems in the pseudoknot structure is labeled. The slippery sequence is underlined. The stop codon in the 0 frame is labeled in red. A13533, which varies from a cytosine in SARS-CoV, is labeled in blue. (C) Validation of the frameshift reporter. Mutating the slippery sequence (ΔSS), disrupting Stem 1 (ΔStem 1), or adding an in-frame stop codon upstream of pseudoknot eliminates frameshifting. **, p<0.01, two-sided t tests. (D) Representative images of cells transfected with mCherry-FSECoV-2-GFP(0), mCherry-FSECoV-2-GFP(−1), or mCherry-GFP. (E) Distributions of GFP/Cherry intensity ratios of individual cells transfected with mCherry-FSECoV-2-GFP(-1) (black), mCherry-FSECoV-2-GFP(0) (red), or mCherry-GFP (green). (F) Schematic illustration of high-throughput compound screen procedure. (G) Ranked effects of 4,434 tested compounds on mCherry-FSECoV-2-GFP(−1) frameshift efficiency.…

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  Figure 2. Merafloxacin specifically inhibits −1 PRF of beta coronaviruses. (A) Ranked effects of 4,434 tested compounds on mCherry-FSECoV-2-GFP(-1) frameshift efficiency. Fluoroquinolone compounds are labeled in red. (B) Chemical structure of merafloxacin. (C-F) Dose-dependent inhibition of SARS-CoV-2 (C), SARS-CoV (D), hCoVO-C43 (E), and hCoV-HKU1 (F) −1 PRF by merafloxacin, showing IC50 values. (G) Effects of merafloxacin on −1 PRF of a variety of FSEs. *, p<0.05; **, p<0.01, two-sided paired ratio t tests.

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  Figure 3. Frameshift inhibition by merafloxacin is robust to mutations. (A) Positions of the naturally occurring C-to-U mutations, labeled in blue. (B) Effects of merafloxacin on −1 PRF of each FSE natural variant. ***, p<0.001, two-sided ratio t tests. (C) Positions of the structure-perturbing, synonymous mutations. Each group of mutations is labeled in the same color. (D) Effects of merafloxacin on −1 PRF of each FSE mutant. *, p<0.05; **, p<0.01; ***, p<0.001, two-sided paired ratio t tests.

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  Figure 4. Merafloxacin inhibits SARS-CoV-2 replication in Vero E6 cells. (A) Viral yields after 48 hours of merafloxacin treatment. PFU, plaque forming unit. Detection limit is indicated by dashed line. (B) Effects of merafloxacin on relative viral growth, based on data from (A). EC50 and EC90 values are shown. (C) Relationship between viral titers and relative −1 PRF efficiency, using data from (A) and Figure 2C.