FIG 6.

Bat and guinea pig DPP4 share the same glycosylation site downstream of the site identified to be important in mDPP4 (Fig. 2B). (A) Removal of the glycosylation site from bDPP4 showed no decrease in infection, while removal of glycosylation from gpDPP4 resulted in no increase in infection. (B) Western blot analysis of bDPP4 and gpDPP4 and their respective glycosylation knockout mutants for DPP4 and β-actin expression. Successful glycosylation knockout is indicated by a downward shift of ∼2.5 kDa. (C) DPP4 and mutant variants are expressed on the surface of cells, visible by immunofluorescence. Cells were transfected with each DPP4 ortholog, fixed, and probed with primary goat anti-DPP4 polyclonal antibody (R&D Systems) at 1:50 and secondary donkey anti-goat Alexa Fluor 488 (Life Technologies) at 1:500. Cells were imaged at a magnification of ×20 for DAPI (300-ms exposure) and DPP4 (1.5-s exposure). (D) Fluorescent cell counts of MERS-CoV infection utilizing various DPP4 orthologs. Cells were infected at an MOI of 0.1, and numbers of infected cells were counted at 72 hpi. Each DPP4 ortholog was measured in triplicate. hDPP4, bDPP4, and bDPP4 −gly had levels of infection that are significantly higher than those seen in the absence of DPP4 (P < 0.05 [Student's t test]). gpDPP4 and gpDPP4 −gly infection levels were not significantly different from those seen in the absence of DPP4. Error bars indicate mean values ± 1 standard deviation.