Middle East respiratory syndrome coronavirus (MERS-CoV) internalization does not rely on DPP4 cytoplasmic tail signaling

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) infects respiratory epithelial cells in humans and camels by binding to dipeptidyl peptidase 4 (DPP4) as its entry receptor. DPP4 is a multifunctional type II membrane protein with a long ectodomain and a short six-amino-acid (aa) cytoplasmic tail. MERS-CoV is known to bind to the ectodomain of DPP4 to gain entry into the host cell. However, the role of the cytoplasmic tail in the entry process remains unclear. Here, we show that mutating or deleting individual aa residues or the entire cytoplasmic tail of DPP4 (ΔcytDPP4) does not completely prevent DPP4 from being inserted into the membrane or from allowing the binding of the MERS-CoV spike protein and pseudovirus infection. Although two mutants, ΔcytDPP4, and a single aa deleted DPP4 (ΔK6DPP4) displayed less surface presentation than wtDPP4, the spike protein could still bind and localize on different DPP4 mutants. The reduced surface expression of ΔK6DPP4 might be due to the extended transmembrane domain, which is altered by the hydrophobic tryptophan (W) residue adjacent to the deleted K6. Furthermore, HEK293T cells transiently expressing DPP4 mutants were permeable to MERS-CoV pseudovirus infection. Not only transiently expressing cells but also cells stably expressing the ΔcytDPP4 mutant were susceptible to MERS-CoV pseudoviral infection, indicating that the DPP4 cytoplasmic tail is not required for MERS-CoV entry. Overall, these data suggest that, although MERS-CoV binds to DPP4, other host factors may need to interact with DPP4 or the spike protein to trigger internalization.

Fig. 1. MERS-CoV entry is mediated by two distinct entry pathways.

a Representative brightfield fluorescence image and a corresponding graph showing a reduction in the percentage of infection of MERS-PV-GFP in Huh7 cells upon treatment with MERS-CoV spike polyclonal antibody, compared to the mock-treated group (b) and (c) Bar graph showing the percentage of neutralization of MERS-CoV PV infection in Huh7 cells and HEK293T cells transiently expressing DPP4, respectively d Relative percentage difference in MERS-CoV PV infection on HEK293T cells transiently expressing either pcDNA, DPP4 alone, TMPRSS2 alone or DPP4 + TMPRSS2 both (e–g) Percentage of relative MERS-CoV PV infection in Huh7, Vero and Calu3 respectively on treatment with 100 µM Camostat mesylate, N = 3, bar graphs represent mean ± SEM, ns non-significant, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig. 2. Surface expression and MERS-CoV S1-Fc binding of DPP4 mutants.

a Multiple sequence alignment of DPP4 in different MERS-CoV susceptible and non-susceptible species, marking the putative phosphorylation site at the third amino acid position (letter ‘P’ marked in red). b Schematic representation of different mutations introduced in the cytoplasmic tail of DPP4. c Histograms depicting surface expression of wtDPP4 and its other mutants using flow cytometry. The values in the histogram depicts the percentage of FITC-positive cells. d Percentage difference of different DPP4 mutants surface expression as compared to wtDPP4. e Schematic representation of coronavirus spike protein. f In vitro expression confirmation of recombinant MERS-CoV spike S1-Fc protein using immunocytochemistry, scale bar = 5 µm (g) Molecular mass confirmation of purified recombinant MERS-CoV spike S1-Fc protein through western blot. h Histograms depicting surface binding of MERS-CoV spike S1 on DPP4 and its other mutant expressing cells using flow cytometry, values in the histogram plots indicate the percentage of FITC positive cells. i Percentage difference of MERS-CoV spike S1-Fc protein binding on different DPP4 mutants as compared to wild-type DPP4, N = 3, bar graph represents mean ± SD, ** p < 0.01,*** p < 0.001.

Fig. 3. MERS-CoV spike interaction with mutant DPP4 and permissivity of MERS-CoV pseudotyped virus in DPP4 mutant expressing cells.

a HEK293T cells transiently transfected with wtDPP4, ΔK6DPP4, ΔcytDPP4 or pcDNA showing receptor expression (red) and MERS-CoV spike S1 protein binding (green) upon immunostaining, scale bar = 50 µm (b) and (c) MERS-CoV infection in two non-susceptible cell lines HEK293T and BHK-21 respectively, transiently expressing wtDPP4 and its different mutants, N = 2, bar graphs represent mean ± SEM (HEK293T) and mean ± SD (BHK-21), * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig. 4. Protein expression and colocalization of DPP4 mutants.

a Differential expression of wtDPP4 and its mutants (250 KDa) as analyzed by western blotting, tubulin stained as loading control (55 KDa). b Dual staining of wtDPP4, ΔK6DPP4, ΔcytDPP4, ACE2 or pcDNA (red) and MERS-CoV spike S1 protein (green) to visualize colocalization (yellow), white squares indicate zoomed area, scale bar = 10 µm. c Transmembrane length prediction of wtDPP4 and ΔK6 using the TMHMM v2.0 tool. d Schematic representation of the predicted arrangement of ΔK6DPP4 on the cell membrane in comparison with wtDPP4. e Alphafold3 predicted models of wtDPP4 and ΔK6DPP4 superimposed on each other showing a bend and increase in length of ΔK6DPP4 transmembrane domain as compared to wtDPP4.

Fig. 5. MERS-CoV pseudotyped viruses are permissive in cells stably expressing ΔcytDPP4.

HEK293T stable cell lines expressing either full-length DPP4 ((HEK293TwtDPP4) or cytoplasmic tail-deleted DPP4 (HEK293TΔcytDPP4) were used to assess. a Cell surface expression and MERS-CoV S1 binding (scale bar = 50 µm), b Expression levels of wtDPP4 and ΔcytDPP4 by Western blot, and (c) confirmation of the cytoplasmic tail deletion in ΔcytDPP4 using reverse transcription PCR (RT-PCR) with two distinct primer sets, “Set1” primers were designed to amplify the both full-length DPP4 and ΔcytDPP4 sequence, while “Set2” primers include a forward primer binding site located immediately downstream of the cytoplasmic tail region, allowing amplification only of ΔcytDPP4. d Dual staining of ΔcytDPP4 (red) and SARS-CoV-2 spike S1 protein (green) to visualize colocalization (yellow), white square indicates zoomed area, scale bar = 10 µm. e Dual staining of ΔcytDPP4 (red) and MERS-CoV spike S1 protein (green) to visualize colocalization (yellow), white square indicates zoomed area, scale bar = 10 µm, (f) and (g) Percentage of relative pseudovirus infection in HEK293T^wtDPP4 and HEK293T^ΔcytDPP4 stable cell lines and cells transiently expressing wtDPP4 or ΔcytDPP4 respectively, N = 3, bar graphs represent mean ± SD, ns non-significant, * p < 0.05; ** p < 0.01; *** p < 0.001.