SARS-CoV-2 Spike displays multiple adaptive changes in addition to the furin cleavage site.

Evolution of SARS-CoV-2 from bat sarbecoviruses involved multiple changes in Spike in addition to insertion of the furin cleavage site (FCS). Analysis of the closely related Spike of BANAL-20-52 reveals key adaptations in the SARS-CoV-2 Spike beyond the FCS that occurred prior to the spillover of SARS-CoV-2's immediate progenitor to humans. Bat sarbecoviruses have enteric tropism and spread mostly by the gastrointestinal route. Their Spike proteins predominantly assume the locked form, which is able to resist the low pH of the bat gastrointestinal tract. Initial changes during the SARS-CoV-2 evolutionary pathway included substitutions that expanded the host range of the sarbecovirus progenitor and allowed circulation in nonbat mammals. Adaptation of the SARS-CoV-2 progenitors also involved remodeling of the amino-terminal domain. Respiratory adaptation occurred during circulation in nonbat animals and resulted in greater propensity for Spike to assume open forms that are less compact and more metastable than the locked or closed forms. Substitutions at monomer interfaces in the Spike trimer facilitate the open shift. Like FCS insertion, these substitutions make Spike more susceptible to low pH degradation and could not have occurred in bats. After SARS-CoV-2 spilled over to humans Spikes of the dominant lineage acquired an aspartic acid to glycine substitution at position 614 that further decreases interaction between monomers and promotes opening of the Spike trimer. A multi-stage evolutionary trajectory is also evident during cross-species transmissions of bat sarbecoviruses to pangolins and the first known spillovers of SARS-CoV via the wildlife trade.