Convergent utilization of APN receptor by hedgehog merbecoviruses

Abstract Dipeptidyl peptidase-4 (DPP4) and angiotensin-converting enzyme 2 (ACE2) are well-established receptors for merbecoviruses, yet the receptor usage of merbecoviruses of European and Asian hedgehogs (EriCoVs) remains unknown. Here, by testing hedgehog orthologs of known coronavirus receptors, we identify hedgehog aminopeptidase N (APN) as a functional receptor for various EriCoVs. Analysis of APN orthologs from 139 species reveals that EriCoVs exhibit a restricted host range, primarily utilizing APN from hedgehogs and, to a lesser extent, felids and some other species, shaped by specific molecular determinants at the virus–receptor interface. Cryo-EM analysis of EriCoV–APN complex reveals a novel APN-binding mode that is distinct from those used by alpha- and deltacoronaviruses. Functional assays further demonstrate that transmembrane serine protease 2 (TMPRSS2) enhances spike activation and promotes plasma membrane fusion. Neutralizing antibodies targeting the EriCoV RBD and hedgehog APN were generated and effectively blocked EriCoV pseudovirus entry or amplification in hedgehog-APN expressing human cells or primary hedgehog cells. The structural basis underlying the pan-EriCoV neutralization mediated by the RBD-targeting antibody BD23-0177 was further elucidated by cryo-EM analysis. Together, these findings uncover an unexpected convergent evolution of APN utilization among merbecoviruses, establishing a foundation for risk assessment and the development of targeted countermeasures.