Evasion to NSP1-mediated translation inhibition with NSP1 variants containing mutations in the amino-terminal domain of NSP1. (A) Cartoon depicting the reporter mRNAs used for translation in RRL, they contain the SARS-CoV-2 5′UTR and the first 12 amino-terminal codons of NSP1 fused to the Renilla luciferase coding sequence. The reporter mRNA contains the wild-type SL1 hairpin or mut3 which has a U to A substitution in the loop or the SARS-CoV-1 SL1. (B) Curves representing the average relative activities measured after translation in RRL in the absence or presence of 0.1, 0.2, 0.3, 0.4, or 0.5 µM of wild-type NSP1, (C) Δ12, and (D) P6L/F8V. The averages are obtained from four independent experiments. Standard deviations or translational activity for each transcript are shown and calculated from four independent experiments. ns: nonsignificant; (*) 0.05 > P-value >0.01; (**) 0.01 > P-value >0.001; (***) P-value <0.001; based on Student's t-test. To facilitate comparisons between NSP1 proteins, the results have also been presented according to the hairpin SL1 tested (Supplemental Fig. S9).
