Figure 6. Re-occurrence of amino acids observed in other sarbecoviruses during SARS-CoV-2 evolution.

(A) Sequence alignment shows recently mutated amino acids in SARS-CoV-2 that are found in other ACE2-binding sarbecoviruses⁴⁹. Re-occurring amino acids are highlighted in yellow. Sequence accession codes are provided at the end of each viral strain name. (B) Frequency of amino acids at particular positions on SARS-CoV-2 spike as of October 2024. Data were sourced from nextstrain.org (GISAID data)^14,50. (C) Sequence conservation analysis of ACE2-binding sarbecoviruses excluding SARS-CoV-2. Each row corresponds to the residue in panel B. Amino acids that have re-occurred in the currently dominant SARS-CoV-2 variant KP.3.1.1 are shown in red. The SARS-CoV-2 S31 deletion results in an N-glycosylation site ³⁰NFT, corresponding to the N-glycosylation site ³⁰NSS in some other sarbecoviruses, which is also highlighted here. The sequence conservation is plotted by WebLogo⁵¹. (D) Fold change in hACE2 binding affinity (Δlog10 K_D), entry into 293T cells expressing high ACE2 (functional score), RBD expression (log10(MFI)) for assessment of re-occurring mutations. The average was calculated from all mutations at that site. Green highlights where the effect of the re-occurring mutation is greater than the average, and red highlights where the effect of the re-occurring mutation is less than the average. Data are from previous DMS studies^16,41,42. (See also figure S10).