Coronaviruses often cross species barriers, with receptor binding dictating their host range and zoonotic potential. Merbecoviruses, such as MERS-CoV, typically utilize DPP4 as their receptor, whereas Sarbecoviruses, like SARS-CoV, rely on ACE2. This study explores the receptor usage of four merbecoviruses identified in Vespertilionidae bats: HKU5, BtVs-SC2013, HKU25, and P. khulii-2011. Our findings reveal species-specific binding to bat ACE2: HKU5 binds exclusively to Pipistrellus abramus ACE2, P. khulii-2011 interacts solely with Murina aurata ACE2, BtVs-SC2013 recognizes ACE2 from Murina aurata and Myotis myotis, and HKU25 displays the broadest binding range. Beyond bats, BtVs-SC2013 binds to mink ACE2, while HKU25 interacts with both mink and pangolin ACE2, hinting at potential intermediate hosts for cross-species transmission. We also elucidated the mechanism behind HKU5’s selective binding preference for P. abramus ACE2. Structural analysis and mutagenesis revealed that a carbohydrate attached at position 329 play a crucial role. Introducing the N-glycosylation site into P. abramus ACE2 eliminated binding, while its removal from P. pipistrellus ACE2, combined with two additional mutations, restored it. Moreover, we pinpointed key residues in mink ACE2 essential for binding the receptor-binding domain (RBD) of BtVs-SC2013 and HKU25. These findings illuminate the receptor usage and host specificity of bat merbecoviruses, enhancing our understanding of their potential for cross-species transmission and adaptation.
