FIG 4.

BtCoV/Ii/GD/2014-422 RBD analysis and DPP4-binding assay. (A) Sequence alignment of the partial S1 domains (including all positions with direct interactions with human DPP4) of selected lineage C betacoronaviruses. Asterisks indicate positions with fully conserved residues. Colons indicate positions with strongly conserved residues. Periods indicate positions with weakly conserved residues. Positions that have direct interactions with human DPP4 according to data from MERS-CoV are in gray bars. Residues identical to corresponding MERS-CoV residues are in red. (B) AlphaScreen assay showing the direct binding interactions between the coronavirus spike RBD and hDPP4 or bDPP4. Binding affinity was characterized as AlphaScreen counts. Error bars indicate standard errors of the means (SEM) (*, P < 0.05 by two-tailed t test; n = 3). (C) Dot blot hybridization assay showing the direct binding interactions between the coronavirus spike RBD and hDPP4 or bDPP4. His₈-tagged hDPP4, bDPP4, or hACE2 was dotted and then incubated with each of the Fc-tagged coronavirus RBDs, followed by anti-IgG4 monoclonal antibody detection.