Stringent selection drives convergence toward omicron-like SARS-CoV-2 receptor-binding motifs.

In vitro protein evolution can provide powerful insights into the amino acid sequences that underlie key biological functions. Here, we use this to explore the evolutionary trajectories of the SARS-CoV-2 spike protein receptor-binding motif (RBM) binding the human angiotensin-converting enzyme 2 (ACE2), an essential first step in viral infection. Applying stringent selection pressures starting from the Wuhan or another non-Omicron variant protein-coding sequence results in rapid convergence towards Omicron characteristic mutations and its sub-lineages. Conversely, under mild selection, only some Omicron-like mutations are selected, however at lower frequencies and with incomplete representation. Stringent selection results in fewer, but dominant, non-synonymous mutations mirroring Omicron mutations and their variations within its sub-lineages. Notably, initiating evolution from Omicron itself results in maintenance of Omicron-defining mutations under both conditions. This evolutionary pattern parallels global SARS-CoV-2 mutation trends as well as in silico simulations, emphasizing the critical role of receptor-binding constraints in shaping viral adaptation. Mutations primarily associated with immune evasion are not selected by in vitro evolution. Our findings demonstrate the predictive capacity of in vitro evolution, suggesting Omicron RBM to be the humanized binding motif, emerging from high-stringency selection, superimposed on milder background pressures.