Mouse dipeptidyl peptidase 4 is not a functional receptor for Middle East respiratory syndrome coronavirus infection

Abstract

Human dipeptidyl peptidase 4 (hDPP4) was recently identified as the receptor for Middle East respiratory syndrome coronavirus (MERS-CoV) infection, suggesting that other mammalian DPP4 orthologs may also support infection. We demonstrate that mouse DPP4 cannot support MERS-CoV infection. However, employing mouse DPP4 as a scaffold, we identified two critical amino acids (A288L and T330R) that regulate species specificity in the mouse. This knowledge can support the rational design of a mouse-adapted MERS-CoV for rapid assessment of therapeutics.

Figures

FIG 1

Mouse DPP4 (mDPP4) does not support MERS-CoV infection. HEK 293T cells were transfected with 3 μg of plasmid expressing human DPP4 (hDPP4) or hDPP4-Venus fusion (A) and mDPP4 or mDPP4-venus fusion (B). At ∼20 h posttransfection, cells were infected with rMERS-CoV-red virus at an MOI of 5. Venus fusion proteins were assessed by fluorescence microscopy at 48 h posttransfection. In independent experiments, infection with rMERS-CoV-red virus was assessed for red cells by fluorescence microscopy at ∼18 h postinfection. (C) Western blot analysis demonstrates overexpression of mDPP4 and hDPP4. Extracts were prepared at ∼48 h posttransfection using AV lysis buffer (3), and samples were heat inactivated for 60 min at 90°C for removal from a biosafety level 3 (BSL3) facility and resolved on an 8% SDS-PAGE gel. Blots were probed with primary goat-anti-DPP4 polyclonal antibody (R&D Systems) at 1:1,000 in 1× Tris-buffered saline–Tween (TBST) or goat anti-actin polyclonal antibody (Santa Cruz) and detected with a secondary rabbit anti-goat–horseradish peroxidase (HRP)-conjugated antibody (Sigma) at 1:10,000 in 1× TBST in 5% milk. (D) Western blot analysis of MERS-CoV S and N proteins. Lysates were collected at ∼18 h postinfection and treated as in panel C. Blots were probed with primary mouse polyclonal antiserum at 1:400, raised to S and N proteins as described previously (3), and detected with a secondary goat anti-mouse–HRP (GE Healthcare) at 1:10,000 in 1× TBST in 5% milk.

FIG 2

Blades IV and V from the β-propeller of hDPP4 make mDPP4 permissible to MERS-CoV infection. (A) Vector NTI protein sequence alignment of human (top strand) and mouse (bottom strand) DPP4 molecules. Yellow highlighted regions indicate conserved amino acids, white regions signify amino acids that are functionally different (i.e., hydrophobic and hydrophilic), and green highlighting indicates amino acids that are different but functionally similar (i.e., the threonine and serine are both polar and uncharged). (B) HEK 293T cells were transfected with the indicated DPP4. At ∼20 h posttransfection, cells were infected with rMERS-CoV-red virus at an MOI of 5, and infection was assessed ∼18 h postinfection by fluorescence microscopy. (C) 3D molecular PyMOL software was employed to visualize the mDPP4 structure overlaid onto the hDPP4 structure. The hDPP4 structure was based upon the crystal structure resolved in context with the MERS S RBD (PDB code 4L72). MERS S protein is displayed in red, hDPP4 in yellow, and mDPP4 in blue. The mDPP4 sequence was threaded using the I-TASSER software (11). The expanded view depicts the DPP4 region at the interaction surface. Numbered and highlighted are the specific amino acids chosen for mutation in the mDPP4 protein.

FIG 3

MERS-CoV infection is dependent upon specific amino acids in DPP4. (A) Vector NTI protein sequence alignment of hDPP4 (top strand) with chDPP4 (middle strand) and mDPP4 (bottom strand) indicating positions of introduced human mutations with red arrows. (B) HEK 293T cells were transfected with the indicated DPP4 molecule. At ∼20 h posttransfection, cells were infected with rMERS-CoV-red virus at MOI of 1, and infection was assessed ∼18 h postinfection by fluorescence microscopy. (C) In an independent experiment, cells overexpressing the indicated DPP4 constructs were infected with rMERS-CoV-red virus at MOI of 0.1, 0.01, and 0.001 on six-well plates. At ∼18 h postinfection, cells were scored at the following MOI: no DPP4 and mDPP4, 0.1; chDPP4 P282T A288L R289I T330R V340I [“chDPP4 (5 mutations)”] and chDPP4 A288L T330R, 0.01; and hDPP4, 0.001. Values were normalized to an MOI of 0.1 and expressed as relative infection at 0.1. Human DPP4, chDPP4 P282T A288L R289I T330R V340I, and chDPP4 A288L T330R showed a significant increase in infection over mDPP4 (*, P < 0.05, Student's t test). (D) Western blots demonstrating overexpression of hDPP4, mDPP4, and each chDPP4 molecule, N protein of infected cells, and β-actin as a loading control. Western blots were prepared and probed as described in Fig. 1C and D.

FIG 4

Human and chimeric DPP4 molecules can support MERS-CoV infection in hamster and mouse cells. (A) Baby hamster kidney 21 (BHK-21) cells were electroporated with the indicated DPP4 molecules. At ∼20 h posttransfection, cells were seeded in 6-well plates and infected with rMERS-CoV-red at an MOI of 2 at 24 h posttransfection. (B) Mouse NIH 3T3 cells were transfected using Nucleofection (according to the Amaxa procedure) with the indicated DPP4 molecules. Cells were seeded into 12-well plates and infected with rMERS-CoV-red at an MOI of ∼4 at 24 h post-Nucleofection. All infections were assessed at 24 h postinfection by fluorescence microscopy.