Fig. 5. Mutations within the RBD that confer specificity toward WT ACE2 are rare.

(A) The SARS-CoV-2 RBD is colored by specificity score [the difference between the conservation scores for cells collected in the sACE2₂(WT)- and sACE2₂.v2.4-specific gates]. Residues that are hotspots for mutations with increased specificity toward sACE2₂(WT) are blue or toward sACE2₂.v2.4 are purple. The contacting surface of ACE2 is shown as a green ribbon, with sites of mutations in sACE2₂.v2.4 labeled and shown as green spheres. (B) Log₂ enrichment ratios for mutations in S expressed by cell populations collected in the sACE2₂(WT)-specific (x axis) and sACE2₂.v2.4-specific (y axis) gates. Data are the mean from two independent sorting experiments. S mutants in blue were predicted to have increased specificity for sACE2₂(WT) and were tested by targeted mutagenesis in fig. S6. S mutants in purple were predicted to have increased specificity for sACE2₂.v2.4 and were tested by targeted mutagenesis in fig. S7. Other nonsynonymous mutations are black. Nonsense mutations are red. (C) WT myc-S (gray) and three variants, Y449K (purple), N501W (light blue), and N501Y (dark blue), were expressed in Expi293F cells and tested by flow cytometry for binding to sACE2₂(WT)-8h (dashed lines) or sACE2₂.v2.4-8h (solid lines). MFU, mean fluorescence units.