ACE2 binding is an ancestral and evolvable trait of sarbecoviruses.

Two different sarbecoviruses have caused major human outbreaks in the past two decades. Both of these sarbecoviruses, SARS-CoV-1 and SARS-CoV-2, engage ACE2 through the spike receptor-binding domain. However, binding to ACE2 orthologues of humans, bats and other species has been observed only sporadically among the broader diversity of bat sarbecoviruses. Here we use high-throughput assays to trace the evolutionary history of ACE2 binding across a diverse range of sarbecoviruses and ACE2 orthologues. We find that ACE2 binding is an ancestral trait of sarbecovirus receptor-binding domains that has subsequently been lost in some clades. Furthermore, we reveal that bat sarbecoviruses from outside Asia can bind to ACE2. Moreover, ACE2 binding is highly evolvable-for many sarbecovirus receptor-binding domains, there are single amino-acid mutations that enable binding to new ACE2 orthologues. However, the effects of individual mutations can differ considerably between viruses, as shown by the N501Y mutation, which enhances the human ACE2-binding affinity of several SARS-CoV-2 variants of concern but substantially decreases it for SARS-CoV-1. Our results point to the deep ancestral origin and evolutionary plasticity of ACE2 binding, broadening the range of sarbecoviruses that should be considered to have spillover potential.