Sarbecovirus Rc-o319 S-Protein Structures Reveal Highly Specialized Adaptation to Rhinolophus cornutus ACE2

Bat sarbecoviruses often exhibit species-dependent ACE2 specificity. Understanding the determinants of receptor specificity enables better assessment of the cross-species transmission potential of sarbecoviruses. Here, we characterize the S-protein of Rc-o319, a sarbecovirus identified in Japanese Rhinolophus cornutus bats. Featuring an unusual 9-amino-acid deletion within its receptor binding motif (RBM), Rc-o319 S-protein utilizes its cognate R. cornutus ACE2 (bACE2R.cor) but not human ACE2 (hACE2), demonstrating highly restricted receptor specificity. Cryo-EM structures reveal two locked prefusion conformations of the Rc-o319 S-trimer and define a novel type-1b receptor-binding domain (RBD), featuring a distinct beta-loop (BL) within the RBM due to the RBM-deletion. The Rc-o319-RBD:bACE2R.cor complex structure reveals unique interactions mediated by the specialized BL and RBM-loop of Rc-o319-RBD and by a bACE2R.corglycan. Structure-guided mutagenesis demonstrates that changes in BL and RBM-loop within the Rc-o319 S-RBD must occur simultaneously to allow medium-to-high-affinity hACE2 binding. Comparative assays further show that the bACE2R.cor receptor supports only a subset of sarbecoviruses, highlighting its restricted sarbecovirus compatibility. Our findings establish the Rc-o319 S-protein as a structurally and functionally specialized adaptation to R. cornutus ACE2 and identify the structural constraints limiting its cross-species transmission potential. Author SummarySARS-related viruses are widely found in horseshoe bats. Some can bind the human ACE2 receptor with variable affinities, whereas others bind only bat ACE2 receptors. Understanding the basis for this difference is critical for assessing spillover risk. We studied the spike protein of a bat sarbecovirus, Rc-o319, isolated from Japanese Rhinolophus cornutus horseshoe bats. Although Rc-o319 is genetically related to SARS-CoV-2, it is unable to bind the human ACE2 receptor. Structural analyses and functional experiments revealed that the Rc-o319 spike protein possesses a distinct receptor-binding motif (RBM). This RBM features a specialized "beta-loop" that replaces the "large-loop" found in human-infecting sarbecoviruses. The beta-loop enables high-affinity binding to its cognate Rhinolophus cornutus bat ACE2 receptor while simultaneously preventing optimal interaction with human ACE2. We further found that acquisition of high affinity for the human ACE2 receptor would require coordinated changes across multiple regions of the RBM, including changing the beta-loop into a large-loop structure. Together, our findings demonstrate that the Rc-o319 spike protein is highly adapted to its cognate bat ACE2 receptor and faces substantial structural constraints that limit its ability to switch to binding human ACE2.