Adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the Middle East respiratory syndrome coronavirus

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) replicates in cells of different species using dipeptidyl peptidase 4 (DPP4) as a functional receptor. Here we show the resistance of ferrets to MERS-CoV infection and inability of ferret DDP4 to bind MERS-CoV. Site-directed mutagenesis of amino acids variable in ferret DPP4 thus revealed the functional human DPP4 virus binding site. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection.

FIG 1

Ferrets resist MERS-CoV infection. (A and B) MERS-CoV in throat (A) and nose (B) swabs of ferrets (n = 4) inoculated with MERS-CoV was tested for the presence of human CoV (HCoV-EMC) RNA using a TaqMan assay. Ct, threshold cycle. (C and D) Fluorescence-activated cell sorter (FACS) analyses of DPP4 staining or S1-Fc binding on ferret kidney cells incubated with either goat anti-DPP4 polyclonal serum or S1-Fc (5 μg/ml) followed by incubation with fluorescein isothiocyanate (FITC)-labeled rabbit anti-goat IgG antibody or FITC-labeled goat anti-human IgG, respectively (red lines). Normal goat serum, feline CoV S1-Fc protein (blue lines), and mock-incubated cells (gray shading) were used as controls. (E) MERS-CoV infection of primary ferret kidney cells transfected with a control plasmid or with a plasmid encoding hDPP4, stained for DPP4, S1 binding, and MERS-CoV as described previously (13).

FIG 2

Ferret DPP4 does not bind MERS-CoV spike protein. (A) Different plasmids encoding full-length human DPP4, ferret DPP4, or human-ferret DPP4 chimeras (human-ferret-human and ferret-human-ferret [HFH and FHF, respectively]) were constructed. (B) DPP4 expression and S1 binding to cells transfected with different DPP4 constructs as determined by FACS analysis. Each experiment was conducted in triplicate, and the results shown are representative of two different experiments. (C) MERS-CoV RNA levels in supernatants of DPP4-transfected cells infected with MERS-CoV at 2 and 20 h after infection using a TaqMan assay. Results representative of three different experiments are shown and are expressed as genome equivalents (GE; half-maximal tissue culture infectious dose [TCID₅₀] per ml).

FIG 3

Characterization of the functional MERS-CoV DPP4 receptor S1 binding site. (A) Amino acid sequence alignment of DPP4 loops 4 and 5 from different species. (B) S1 binding assy. Different hDPP4 mutant-transfected cells were harvested and incubated with 5 μg of MERS-CoV-S1-Fc protein, a second step was performed with FITC-labeled anti-human IgG, and cells were analyzed by FACS. The error bars indicate standard errors of the means. (C) MERS-CoV infection of cells transfected with different hDPP4 constructs. Data in panel B and C were corrected for DPP4 expression of the different constructs (one-way analysis of variance [ANOVA] test; *, P < 0.05; **, P < 0.01; n = 3 per group). (D and E) Phylogenetic tree based on the complete DPP4 (D) and a DPP4 fragment containing the S1 binding region (residues 246 to 504) (E) of 13 different species. Sequence alignment was performed by using ClustalW in the MEGA5.0 software package (www.megasoftware.net), and the trees were constructed by using the neighbor-joining method with P distances (gap/missing data treatment; complete deletion) and 1,000 bootstrap replicates as in MEGA version 5.0.

FIG 4

Natural antagonist ADA blocks MERS-CoV binding and infection. (A) hDPP4 plasmid-transfected MDCK cells were preincubated with 5 μg of recombinant ADA for 1 h, after which the cells were analyzed for S1 binding, DPP4 staining, and MERS-CoV infection. On hDPP4-transfected cells, ADA blocks S1 binding and MERS-CoV infection despite the expression of DPP4, visualized by fluorescent staining for DPP4, S1, and MERS-CoV, respectively. (B) Recombinant human ADA (R&D Systems)-preincubated Huh7 cells were infected with MERS-CoV for 8 h and processed. ADA (open bars) but not recombinant human angiotensin-converting enzyme 2 (rACE2) (closed bars) dose-dependently blocked MERS-CoV infection of Huh7 cells. Results representative of three different experiments are shown (one-way ANOVA test; *, P < 0.05; n = 3 per group).