Fig. 5: Molecular determinants of HKU5-ACE2-mediated entry.
From: HKU5 bat merbecoviruses engage bat and mink ACE2 as entry receptors
a Schematic of the ACE2 gene highlighting substituted residues and domains. Positions in gray had minimal effect on HKU5 entry, while substitutions that reduced entry are shown in red. b Wild-type P. abramus ACE2, individual, and combinatorial ACE2 mutants were transiently transfected into BHK-21 cells and then infected with VSV-HKU5spike-RLuc or VSV-SARS-CoVspike-RLuc. c We generated two patches of substitutions (1, 2) on the HKU5RBD and tested each for infectivity on BHK-21 cells stably expressing P. abramus ACE2 (Patch 1: Y507A/Y544Q/Y557SRBD; patch 2: E510K/Y512S/Y517S/Y521E/Y523ARBD. Patches 1 and 2 of the HKU5RBD were necessary for P. abramus ACE2 utilization, while individual substitutions Y507ARBD and E510KRBD were sufficient to block infection. d We generated four patches of substitutions (3, 4, 5, 6) on the HKU5RBD (orange) and tested each for infectivity on BHK-21 cells stably expressing P. abramus ACE2 (green). Patch 3: S457P/D459S/Y463D/A471PRBD; patch 4: T509N/E510K/Y512S/T514L/S515L/A516F/Y517D/G518D/K519R/Y521ERBD; patch 5: 542-548→EDGDYYRKQLSPLEGRBD; patch 6: T553A/T555S/Y557S/I558T/Y559VRBD). HKU5 patches 4, 5, and 6 were required for P. abramus ACE2 use, with individual substitutions K519RRBD and Q545PRBD sufficient to block infection. Dots represent the mean from each of three independent experiments, each done in technical triplicate. Statistical analyses were performed using two-tailed unpaired Student’s t-tests and one-way ANOVA. Data are mean ± s.e.m. b *D90N-I92T-I93V: p = 0.0426, D90N: p = 0.0317, **V30D: p = 0.0089, **I93V: p = 0.0094, **R328E: p = 0.0020, ****p < 0.0001. c *p = 0.0445, ***p = 0005, ****p < 0.0001. d *Y463D: p = 0.0294, *T514L: p = 0.0473, *S515L: p = 0.0176, *A516F: p = 0.0158, **Patch 1: p = 0.0060, **K543D: p = 0.0047, ****p < 0.0001. e HKU5RBD patches 3, 4, 5, and 6. Residue positions for each set of substitutions are shown in stick and dot representations. K519RBD and Q545RBD, which showed infection-blocking effects when mutated, are labeled along with other residues for positional reference. Source data are provided as a Source Data file.
