Deep mutational scanning of recent SARS-CoV-2 variants highlights changing amino acid preferences within epistatic hotspot residues.

Deep mutational scans across receptor-binding domains (RBDs) of diverging SARS-CoV-2 variants reveal ongoing changes to the effects of mutations, a phenomenon known as epistasis. Careful accounting for these altered mutational effects is important in viral surveillance and forecasting, and more broadly, for understanding the impacts of epistasis on real-world viral evolutionary trajectories. Using a yeast-display RBD deep mutational scanning (DMS) platform, we measure the impacts of virtually all single amino acid mutations and single-residue deletions in the Omicron KP.3.1.1 and LP.8.1 RBDs on folded RBD expression and binding affinity for the human ACE2 receptor. Our comprehensive maps reveal patterns of evolutionary accessibility and constraint at single-residue resolution and, when compared to prior datasets, highlight sites whose amino acid preferences continue to change across viral variants. Notably, sites 455, 456, and 493 - which have exhibited repeated substitutions and epistatic dependencies across Omicron subvariants going back to BA.1 - again demonstrate altered patterns of mutational accessibility and constraint. Therefore, it appears that these hotspots of repeated RBD evolution have not yet converged on fixed amino acid solutions but instead remain sites of ongoing epistatic reconfiguration. We compare our measurements of direct RBD:ACE2 affinity with recently published measurements of mutation impacts on ACE2 binding in the full quaternary spike context, which also integrates the effects of spike conformational dynamics; our analysis uncovers mutations like H505W that could favor adoption of the down/closed RBD conformation as a viral strategy for future antigenic evolution.