Summary Phylogenetically distant coronaviruses have evolved to use ACE2 as their common receptors, including NL63 and many Severe acute respiratory syndrome (SARS) coronavirus-related viruses. We recently reported two Middle East respiratory syndrome coronavirus (MERS-CoV) closely related bat merbecoviruses, NeoCoV and PDF-2180, use Angiotensin-converting enzyme 2 (ACE2) for entry. However, their host range and cross-species transmissibility remain unknown. Here, we characterized their species-specific receptor preference by testing ACE2 orthologs from 49 bats and 53 non-bat mammals. Both viruses exhibited broad receptor recognition spectra and are unable to use ACE2 orthologs from 24 species, mainly Yinpterochiropteran bats. Comparative analyses of bat ACE2 orthologs underscored four crucial host range determinants, all confirmed by subsequent functional assays in human and bat cells. Among them, residue 305, participating in a critical interaction, plays a crucial role in host tropism determination. NeoCoV-T510F, a mutation that enhances human ACE2 recognition, further expanded the potential host range via tighter interaction with an evolutionary conserved hydrophobic pocket. Our results elucidated the molecular basis for the species-specific ACE2 usage of MERS-related viruses across mammals and shed light on their zoonotic risks.

Broad host tropism of ACE2-using MERS-related coronaviruses and determinants restricting viral recognition