FIG 5.

Many amino acid changes are required to make fDPP4 and haDPP4 permissive to MERS-CoV infection. (A) Removing glycosylation alone did not confer permissivity to haDPP4. However, combining three amino acid changes on blade V (starting at residue 289) with the glycosylation knockout mutant on blade IV (N332A) resulted in high levels of MERS-CoV infection. Sequences show the alignment between hDPP4 and haDPP4, with the black boxes indicating the amino acids that were swapped from hDPP4 into haDPP4. (B) Removing glycosylation alone did not confer permissivity to fDPP4. However, introducing a set of amino acid changes on blade V (starting at residue 278) and blade IV (starting at residue 330) allowed fDPP4 to support MERS-CoV infection [fDPP4 (278) (330)]. Sequences show the alignment between hDPP4 and fDPP4, with the black boxes indicating the amino acids that were swapped from hDPP4 into fDPP4. Note that fDPP4 −gly is a negative control and includes only the single point mutation N331A. (C) Western blot analysis of fDPP4 and haDPP4 and designated variants for DPP4 and β-actin expression. Successful glycosylation knockout is indicated by a downward shift of ∼2.5 kDa. (D) Fluorescent cell counts of MERS-CoV infection utilizing DPP4 orthologs. Cells were infected at an MOI of 0.1, and numbers of red cells were counted at 72 hpi. Each DPP4 ortholog was measured in triplicate. hDPP4, fDPP4 (278) (330), and haDPP4 (289), −gly had levels of infection that were significantly greater than those seen in the absence of DPP4 (P < 0.05 [Student's t test]). fDPP4 −gly and haDPP4 −gly infection levels were not significantly different from those seen in the absence of DPP4. Error bars indicate mean values ± 1 standard deviation.