Molecular Insights into Species-Specific ACE2 Recognition of Coronavirus HKU5

Coronaviruses represent a significant zoonotic threat, with host adaptation serving as a pivotal determinant of cross-species transmission. The bat-derived β-coronavirus HKU5 utilizes its spike (S) protein for receptor recognition and viral entry. Here, we report the cryo-electron microscopy (cryo-EM) structure of the HKU5 S protein in a closed conformation. Two fatty acids are found in each protomer of the HKU5 S protein, which stabilize the S protein in the closed conformation. Furthermore, we solve the structure of the HKU5 receptor-binding domain (RBD) in complex with the peptidase domain (PD) of Pipistrellus abramus angiotensin-converting enzyme 2 (ACE2), uncovering a unique binding mode distinct from other coronaviruses that use ACE2 as their receptor. Evolutionary and functional analyses indicate that mutations in the RBD can modulate receptor binding, while conservation and structural modeling suggest that HKU5 has the potential to cross the species barrier. Notably, we identify ACE2 orthologs in avian species, such as Pitta sordida , that support stable HKU5 RBD binding and interaction. Our functional assays, including pseudovirus entry and cell–cell fusion experiments, demonstrate that HKU5 can exploit ACE2 orthologs across species, providing molecular insights into its host adaptation and underscoring the importance of surveillance for this virus and its zoonotic risk.