Extended Data Fig. 2: Deep mutational scanning of the ACE2-binding ability of the SARS-CoV-2 Spike.
a) Representative images showing the syncytium-forming potential of Omicron Spike variants. The fusion-defective HexaPro mutant and the Delta variant were included for comparison. b) Heatmap depicting how all single mutations affect the ACE2 binding of Spike’s FPPR. Squares are colored by mutational effect according to scale bars on the right, with red and purple indicating ACE2 binding-enhancing and inhibiting substitutions, respectively. The mutations with no measurement are in gray cross. The SARS-CoV-2 amino acid is indicated with a black dot. Stop codon is indicated with *. The FC value represents the ACE2 binding ability for each of the single mutations in the Spike FPPR library. It is calculated as the fold change comparing each variant’s relative abundance in FACS-sorted (that is, AF405-positive) ACE2-bound cell pool versus the unsorted cell pool and is normalized to wild-type. High reproducibility of the profiling result was detected between two biological replicates. c) Correlation of the syncytium-forming potential and the ability of ACE2 binding of all single mutations of Spike’s FPPR. Mutants validated are highlighted and labeled. R is the Pearson correlation coefficient. d) Cell surface staining of Spike FPPR variants in HEK293T sender cells. Anti-Spike antibodies against its S2 and S1 subunit were used to evaluate the total surface expression of Spike protein and the level of S1-cleaved Spike on cell surface, respectively. Ctr represents the control cells without Spike expression.
