Sarbecovirus RBD indels and specific residues dictating multi-species ACE2 adaptiveness

Our comprehensive understanding of the multi-species ACE2 adaptiveness of sarbecoviruses remains elusive, particularly for those with various receptor binding motif (RBM) insertions/deletions (indels). Here, we analyzed RBM sequences from 268 sarbecoviruses categorized into four RBM indel types. We examined the ability of 20 representative sarbecovirus Spike glycoproteins (S) and derivatives in utilizing ACE2 from various bats and several other mammalian species. We reveal that sarbecoviruses with long RBMs (type-I) can achieve broad ACE2 tropism, whereas viruses with single deletions in Region 1 (type-II) or Region 2 (type-III) exhibit narrower ACE2 tropism. Sarbecoviruses with double region deletions (type-IV) completely lost ACE2 usage, which is restricted by clade-specific residues within and outside RBM. Lastly, we propose the evolution of sarbecovirus RBM indels and illustrate how loop lengths, disulfide, and residue determinants shape multi-species ACE2 adaptiveness. This study provides profound insights into the mechanisms governing ACE2 usage and spillover risks of sarbecoviruses.