Deep mutational scans of XBB.1.5 and BQ.1.1 reveal ongoing epistatic drift during SARS-CoV-2 evolution.

Substitutions that fix between SARS-CoV-2 variants can transform the mutational landscape of future evolution via epistasis. For example, large epistatic shifts in mutational effects caused by N501Y underlied the original emergence of Omicron variants, but whether such large epistatic saltations continue to define ongoing SARS-CoV-2 evolution remains unclear. We conducted deep mutational scans to measure the impacts of all single amino acid mutations and single-codon deletions in the spike receptor-binding domain (RBD) on ACE2-binding affinity and protein expression in the recent Omicron BQ.1.1 and XBB.1.5 variants, and we compared mutational patterns to earlier viral strains that we have previously profiled. As with previous RBD deep mutational scans, we find many mutations that are tolerated or even enhance binding to ACE2 receptor. The tolerance of sites to single-codon deletion largely conforms with tolerance to amino acid mutation. Though deletions in the RBD have not yet been seen in dominant lineages, we observe many tolerated deletions including at positions that exhibit indel variation across broader sarbecovirus evolution and in emerging SARS-CoV-2 variants of interest, most notably the well-tolerated Δ483 deletion in BA.2.86. The substitutions that distinguish recent viral variants have not induced as dramatic of epistatic perturbations as N501Y, but we identify ongoing epistatic drift in SARS-CoV-2 variants, including interaction between R493Q reversions and mutations at positions 453, 455, and 456, including mutations like F456L that define the newly emerging EG.5 lineage. Our results highlight ongoing drift in the effects of mutations due to epistasis, which may continue to direct SARS-CoV-2 evolution into new regions of sequence space.