Fig. 2. Variable active site residues elicit differential resistance to nirmatrelvir and ensitrelvir.

(A) Alignment of coronavirus M^pro amino acid sequences spanning residues 41–54 and 163–174 (based on SARS2 M^pro residue position).

(B) Structure of SARS2 M^pro and inhibitors highlighting variable residues that can be mutated (PDB: 7SI9 and 7VU6 for nirmatrelvir and ensitrelvir, respectively).

(C) Relative frequency of amino acid changes at tested variable residues, excluding the respective WT residue (open circle), in SARS2 genomes (1-July-2022, GISAID database).

(D) Relative luminescence of cells expressing Src-M^pro-Tat-fLuc variants in the absence of inhibitor.

(E-F) Dose-response curves of variants using the live cell Src-M^pro-Tat-fLuc assay with 4-fold serial dilution of inhibitor beginning at 10 μM (data are mean +/− SD of biologically independent triplicate experiments).