Bat-infecting merbecovirus HKU5-CoV lineage 2 can use human ACE2 as a cell entry receptor

Merbecoviruses comprise four viral species with remarkable genetic diversity: MERS-related coronavirus, Tylonycteris bat coronavirus HKU4, Pipistrellus bat coronavirus HKU5, and Hedgehog coronavirus 1. However, the potential human spillover risk of animal merbecoviruses remains to be investigated. Here, we reported the discovery of HKU5-CoV lineage 2 (HKU5-CoV-2) in bats that efficiently utilize human angiotensin-converting enzyme 2 (ACE2) as a functional receptor and exhibits a broad host tropism. Cryo-EM analysis of HKU5-CoV-2 receptor-binding domain (RBD) and human ACE2 complex revealed an entirely distinct binding mode compared with other ACE2-utilizing merbecoviruses with RBD footprint largely shared with ACE2-using sarbecoviruses and NL63. Structural and functional analyses indicate that HKU5-CoV-2 has a better adaptation to human ACE2 than lineage 1 HKU5-CoV. Authentic HKU5-CoV-2 infected human ACE2-expressing cell lines and human respiratory and enteric organoids. This study reveals a distinct lineage of HKU5-CoVs in bats that efficiently use human ACE2 and underscores their potential zoonotic risk.