Structural and phenotypic plasticity of the RBD loop2 region is a key determinant for HKU5r-CoVs’ emergence in mink

The emergence of novel coronaviruses from animal reservoirs continues to pose significant zoonotic threats. Here, we investigate the evolutionary origins of a recently reported mink-derived HKU5-related coronavirus (nvHKU5r-CoV), as well as the virus’s structural and functional properties. Phylogenetic and recombination analyses reveal that nvHKU5r-CoV originated from bat HKU5-like viruses circulating in southeastern China. We characterize the spike loop2 region as a critical determinant of ACE2 receptor specificity and show that the bat merbecovirus with the closest loop2 sequence to nvHKU5r-CoV could already utilize mink ACE2. Targeted mutagenesis demonstrates that a single amino acid substitution (R548S) further enables robust entry via human ACE2, highlighting the zoonotic potential of HKU5r-CoVs. Molecular dating suggests prolonged period of circulation in bats prior to this virus’s transmission to mink, and viral entry assays using pseudovirus infectivity and full-length replication competent infectious clones confirm replication in mink ACE2-expressing cells. Using AlphaFold3, we predicted spike-ACE2 binding interfaces consistent with our experimental infectivity results, and instrumental in interpreting the structural basis of these interactions. These findings emphasize the evolutionary plasticity of HKU5r-CoV RBDs and the role of fur farming as potential hotspots for coronavirus emergence.