Glycosylation of mouse DPP4 plays a role in inhibiting Middle East respiratory syndrome coronavirus infection

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes dipeptidyl peptidase 4 (DPP4) as an entry receptor. Mouse DPP4 (mDPP4) does not support MERS-CoV entry; however, changes at positions 288 and 330 can confer permissivity. Position 330 changes the charge and glycosylation state of mDPP4. We show that glycosylation is a major factor impacting DPP4 receptor function. These results provide insight into DPP4 species-specific differences impacting MERS-CoV host range and may inform MERS-CoV mouse model development.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

Is charge or glycosylation important for mediating mouse DPP4 permissivity? (A) MEGA6 protein sequence alignment of DPP4 for various permissive (human, camel, and bat) and nonpermissive (mouse, ferret, hamster, and guinea pig) species, visualized in GeneDoc. Residue numbers are relative to mDPP4. The mutation T330R in mDPP4 introduces a conserved positive charge for permissive hosts but also knocks out a glycosylation site. NCBI accession numbers are as follows: human, NP_001926.2; camel, AIG55259; bat, AGF80256.1; mouse, NP_034204.1; ferret, ABC72084.1; hamster, AIG55262.1; guinea pig, XP_003478612.2. (B) The downward shift in the mDPP4 T330R band is consistent with the removal of glycosylation. Western blot protocol followed that described by Cockrell et al. (4). (C) Structure of hDPP4 (yellow) complexed with the MERS-RBD (red) (PDB code 4L72) visualized using PyMOL Molecular Graphics System, version 1.6.0.0 (Schrodinger, LLC). mDPP4 (blue), threaded through i-TASSER (13), is overlaid to show the key mutations: A288L, T330R, and N328A. Blue indicates wild-type mDPP4 residues, while orange indicates the human amino acid identity. (D) DPP4 constructs used. Whether they are glycosylated at residue 328 and whether the conserved positive arginine is present at residue 330 (numbered relative to mDPP4) are also shown.

FIG 2

Glycosylation can act to dramatically reduce infection by MERS-CoV. (A) HEK 293T cells were transfected with each DPP4 construct and infected with rMERS-CoV-RFP at a multiplicity of infection (MOI) of 1 at ∼18 h posttransfection. At ∼24 h postinfection, cells were imaged. (B) Cells were transfected as described for panel A and infected with rMERS-CoV-RFP at the following MOIs: hDPP4 and hDPP4 R336T, 0.001; no DPP4 and hDPP4+gly, 0.1. At 24 h postinfection, cells were counted based on red fluorescence, and values were normalized to an MOI of 0.1. Values represent 3 replicates. All mutants have levels that are statistically greater than those obtained with no DPP4, and all other pairwise comparisons are also statistically significant (*, P < 0.05; Student's t test). (C) Western blot analysis for MERS nucleocapsid (N) protein, DPP4, and actin as a loading control. The Western blot protocol followed that described by Cockrell et al. (4).

FIG 3

DPP4 and mutant variants are expressed on the surfaces of cells, visible by immunofluorescence. Cells were transfected as described in the legend to Fig. 2A, fixed, and probed with primary goat-anti-DPP4 polyclonal antibody (R&D Systems) at 1:50 and secondary donkey anti-goat IgG–Alexa Fluor 488 (Life Technologies) at 1:500. Cells were imaged at 40× for DAPI (30-ms exposure) and DPP4 (160-ms exposure).

FIG 4

Glycosylation, rather than charge, is a key determinant of mouse DPP4 permissivity to MERS-CoV. (A) Cells were transfected and infected following the protocol detailed in the legend to Fig. 2A. Neither mDPP4 N328A nor mDPP4 T330R can confer permissivity to MERS-CoV; however, both result in strong levels of infection when coupled with A288L. (B) Red cell counts were calculated as for Fig. 2B with the following MOIs: hDPP4, 0.001; mDPP4, mDPP4 A288L, mDPP4 N328A, mDPP4 T330R, and no DPP4, 0.1; mDPP4 A288L, N328A and mDPP4 A288L, T330R, 0.01. Values for all DPP4 constructs are significantly greater than those for no DPP4 and mDPP4 (*, P < 0.05; Student's t test) and significantly less than that for hDPP4 (+, P < 0.05; Student's t test); however, the values for mDPP4 A288L, N328A and mDPP4 A288L, T330R are not statistically different from each other (n.s. [not significant], P < 0.05; Student's t test). (C) Western blot analysis for MERS nucleocapsid (N) protein, DPP4, and actin as a loading control. The Western blot protocol followed that described by Cockrell et al. (4).