Position C15 in the stem of SARS-CoV-2 SL1 is necessary to escape NSP1-mediated repression. (A) Sequence of stem 1b (see Fig. 2A) is important for derepressor function of SL1. The role of mutations, disrupting folding or modifying parameters of the stem and bulge in SL1, was tested in the NSP1-reporter assay, as described in Figure 3B. Values are shown as a percentage of luciferase produced in the presence of the vector for each reporter. Values represent means ± SD from at least three experiments. P-values are calculated as in Figure 1. Mutations and resulting predicted structures (mfold, Zuker 2003) are shown above the plots, with mutated residues in red: 7_11del GGUUUinsCCAAA (i.e., replacement of nucleotides 7 to 11 [GGUUU] by CCAAA) unfolding stem 1a, [17_11delGGUUUinsCCAAA; 29_33delAAACCinsUUUGG] restoring stem 1a, [7_11del GGUUUinsCCAAA; 14_17delACCUinsUGGA] unfolding the stem 1, [7_11del GGUUUinsCCAAA; 14_17delACCUinsUGGA; 22_25delAGGUinsUCCA; 29_33delAAACCinsUUUGG] restoring stem 1, [12_16insUAUAU; 34_38AUAUA] elongating stem 1 by 5 bp, [7_11delCCAAA; 29-33del UUUGG] shortening stem 1 by 5 bp, 27_28delACinsU removing bulge. (B) Position 15C in stem 1b is required for SL1 function. Point mutations unfolding stem 1b, as well as compensatory mutations restoring folding, were introduced into SL1-RL reporter and tested in NSP1-mediated repression assay, as described in A. Mutations introduced in stem 1b: 15C > G unfolding; [15C > G; 24G > C] restoring folding; 16C > G unfolding; [16C > G; 23G > C] restoring folding; [14A > U; 16C > G] unfolding; [14A > U; 16C > G; 23G > C; 25U > A] restoring folding. (C) Three cytosine residues in SARS-CoV-2 SL1, one in stem 1b and two in the loop, are crucial to escape NSP1-mediate repression. Functional residues are marked with red arrows. The data are based on reporter assays shown in Figures 3B, 4A,B.
