Experimental evolution of Rc-o319 sarbecovirus spike protein reveals limited ACE2 adaptability

Bat sarbecoviruses include several potentially human-infective viruses, and evaluating their evolvability is therefore important for assessing emergence risks. In particular, the interaction between the viral spike protein and the host ACE2 receptor is critical for cross-species transmissibility. Here, we focused on the spike protein from Rc-o319, a bat sarbecovirus related to SARS-CoV-2 but unable to infect humans. Using a recombinant vesicular stomatitis virus (VSV), we found that Rc-o319 spike protein ACE2 usage was restricted to its natural host, Rhinolophus cornutus, and to mouse ACE2 at low levels, but not to R. sinicus, ferret, cow, or human ACE2 homologues. Experimental evolution passages of the recombinant VSV expressing the Rc-o319 spike enabled us to study spike adaptation without requiring the authentic virus or posing a risk to humans. We found rapid spike optimization for viral entry through mouse ACE2, driven by a single substitution in the receptor-binding motif. In contrast, repeated passaging under strong selective pressure failed to yield variants capable of using human ACE2, indicating substantial evolutionary constraints. Together, our results demonstrate that the Rc-o319 spike can readily optimize interactions with already permissive ACE2 receptors but is limited in its capacity to acquire usage of a new ACE2 homologue, particularly human ACE2, suggesting a low zoonotic risk. However, some evolved spikes appeared to acquire a residual capacity for ACE2-independent viral entry, although this observation remains to be conclusively demonstrated and requires further investigation.