Fig. 6: HKU5 interacts with mink ACE2 in a manner distinct from that of bat ACE2.
From: HKU5 bat merbecoviruses engage bat and mink ACE2 as entry receptors
a Phylogenetic tree of mustelid species for which ACE2 constructs were tested. Shown are the nine amino acid positions that differ between N. vison (susceptible to HKU5) and M. putorius (resistant to HKU5). The figure was created using PhyloPic, an open-access database licensed under Creative Commons. b Wild-type mustelid ACE2 constructs were transiently transfected into BHK-21 cells, and VSV-HKU5-spike-RLuc and VSV-SARS-CoV-spike-RLuc were used to assess entry. N. vison (American mink) and M. erminea (stoat) ACE2, but not the ACE2 of other mustelids, were sufficient for HKU5 entry. SARS-CoV-2 could use all tested mustelid ACE2s. c Wild-type and mutant N. vison ACE2 and M. putorius ACE2s were transiently transfected into BHK-21 cells, and entry was assessed using VSV-HKU5-spike-RLuc and VSV-SARS-CoV-spike-RLuc. N. vison ACE2 residues at A387ACE2 and R548ACE2 (highlighted in red) are necessary for HKU5 use of N. vison ACE2. No individual substitutions were sufficient for HKU5 use of M. putorius ACE2. Data are means from three independent experiments, each done in triplicate. d Key amino acids are mapped onto the P. abramus ACE2 structure. Positions 387ACE2 and 548ACE2 are necessary for HKU5 to utilize mink ACE2. Statistical analyses were performed using two-tailed unpaired Student’s t-tests and one-way ANOVA. Data are mean ± s.e.m. b *M. erminea-HKU5: p = 0.0412, **N. vison ACE2-HKU5: p = 0.0032, *A. collaris ACE2-SARS-CoV-2: p = 0.0109, *M. nigripes-ACE2: p = 0.0172, **M. meles ACE2-SARS-CoV-2: p = 0.0067, **L. canadensis ACE2-SARS-CoV-2: p = 0.0089, **G. gulo ACE2-SARS-CoV-2: p = 0.0052, **L. lutra ACE2-SARS-CoV-2: p = 0.0016, **M. moschata ACE2-SARS-CoV-2: p = 0.0065, **M. putorius ACE2-SARS-CoV-2: p = 0.0013, **M. lutreola ACE2-SARS-CoV-2: p = 0.0042, ***p = 0.0002. c N. vison ACE2-HKU5: **p = 0.0024, ****p < 0.0001; M. putorius ACE2-HKU5: *p = 0.0207, **p = 0.0061 ***p = 0.0009; N. vison ACE2-SARS-CoV-2: *K309E, p = 0.0245, *K313T, p = 0.0284; *H354R, p = 0.0202; ***p = 0.0002; M. putorius ACE2-SARS-CoV-2: ****p < 0.0001. Source data are provided as a Source Data file.
