Structural Basis for Sarbecovirus Rc-o319 Spike Adaptation to Rhinolophus cornutus Bat ACE2 and Constraints on Switching to Human 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 (bACE2 R.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-4 receptor-binding domain (RBD), featuring a distinct beta-loop (BL) within the RBM due to the RBM-deletion. The Rc-o319-RBD:bACE2 R.cor complex structure reveals unique interactions mediated by the specialized BL and RBM-loop of Rc-o319-RBD and by a bACE2 R.cor glycan. 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 bACE2 R.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.