Middle East respiratory syndrome coronavirus infection mediated by the transmembrane serine protease TMPRSS2

Abstract

The Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host proteases for virus entry into lung cells. In the current study, Vero cells constitutively expressing type II transmembrane serine protease (Vero-TMPRSS2 cells) showed larger syncytia at 18 h after infection with MERS-CoV than after infection with other coronaviruses. Furthermore, the susceptibility of Vero-TMPRSS2 cells to MERS-CoV was 100-fold higher than that of non-TMPRSS2-expressing parental Vero cells. The serine protease inhibitor camostat, which inhibits TMPRSS2 activity, completely blocked syncytium formation but only partially blocked virus entry into Vero-TMPRSS2 cells. Importantly, the coronavirus is thought to enter cells via two distinct pathways, one mediated by TMPRSS2 at the cell surface and the other mediated by cathepsin L in the endosome. Simultaneous treatment with inhibitors of cathepsin L and TMPRSS2 completely blocked virus entry into Vero-TMPRSS2 cells, indicating that MERS-CoV employs both the cell surface and the endosomal pathway to infect Vero-TMPRSS2 cells. In contrast, a single camostat treatment suppressed MERS-CoV entry into human bronchial submucosal gland-derived Calu-3 cells by 10-fold and virus growth by 270-fold, although treatment with both camostat and (23,25)-trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester, a cathepsin inhibitor, or treatment with leupeptin, an inhibitor of cysteine, serine, and threonine peptidases, was no more efficacious than treatment with camostat alone. Further, these inhibitors were not efficacious against MERS-CoV infection of MRC-5 and WI-38 cells, which were derived from lung, but these characters differed from those of mature pneumocytes. These results suggest that a single treatment with camostat is sufficient to block MERS-CoV entry into a well-differentiated lung-derived cell line.

Figures

Fig 1

Syncytium formation induced by proteases in Vero cells infected with MERS-CoV. (A) Vero cells infected with MERS-CoV at an MOI of 0.01 were cultured for 15 h and then treated for 6 h with a variety of proteases, trypsin (1 μg/ml), chymotrypsin (10 μg/ml), elastase (10 μg/ml), thermolysin (1 μg/ml), endoproteinase Arg-C (20 μg/ml), or endoproteinase Lys-C (20 μg/ml). (B) Vero-TMPRSS2 cells were infected with serially diluted MERS-CoV in the absence of exogenous proteases and incubated for 18 h. Cells were fixed with formaldehyde and stained with crystal violet.

Fig 2

Western blot analysis of the MERS-CoV S protein. Culture medium samples and cell lysates were collected from parental Vero cells or Vero-TMPRSS2 cells at 18 h after infection with MERS-CoV. Samples were subjected to SDS-PAGE (3 to 10% gradient gel) and transferred to a nitrocellulose membrane. The viral S protein was detected by using an antipeptide antibody against the VHCR of the MHV-2 S protein, followed by a horseradish peroxidase-conjugated anti-rabbit IgG. MHV-2 was used as the positive control for the anti-VHCR antibody. GAPDH was used as the loading control and was detected by using an anti-GAPDH antibody.

Fig 3

Incorporation of the 45-kDa S protein into virus particles. Culture medium collected from Vero-TMPRSS2 cells at 18 h after infection with 4 log10 TCID50s of MERS-CoV was overlaid onto the top fraction of a sucrose step gradient (20% and 60%) and centrifuged at 28,000 rpm for 1 h. The fractions were drawn from the air-fluid interface (fraction 1), the fluid–20% sucrose interface (fraction 2) and the 20% sucrose–60% sucrose interface (fraction 3) and subjected to Western blot analysis (3 to 10% gradient gel). The viral S protein was detected as described in the legend to Fig. 2.

Fig 4

Induction of fusion from without by MERS-CoV particles. (A) A high-titer sample (MOI = 10) of MERS-CoV or SARS-CoV was adsorbed onto Vero-TMPRSS2 cells on ice for 1 h. The culture medium was then exchanged for fresh, prewarmed medium (37°C). Cells were incubated for 3 or 5 h, fixed, and stained with crystal violet. (B) Vero-TMPRSS2 cells were inoculated with MERS-CoV at a high titer (MOI = 10) or a low titer (MOI = 0.0001) for 1 h on ice and then shifted to 37°C in the presence or absence of cycloheximide (100 μg/ml). Cells were incubated for 5 or 20 h, fixed, and stained with crystal violet. Arrows, fused cells.

Fig 5

Inhibition of syncytium formation and S-protein degradation by camostat. (A) Vero-TMPRSS2 cells were infected with MERS-CoV at an MOI of 0.0001 and incubated at 37°C for 1 h. Serially diluted camostat was then added and incubated with the cells for 18 h. Cells were fixed and stained with crystal violet. Arrow, fused cells. (B) The size of syncytia in the presence or absence of camostat was quantified by counting the number of nuclei in fused cells. Bars and error bars indicate the means and the standard deviations from eight independent syncytia, respectively. ND, syncytia were not detected. (C) Vero-TMPRSS2 cells were infected with MERS-CoV at an MOI of 0.1 and incubated at 37°C for 1 h. Camostat was then added and incubated with the cells for 18 h. Cell lysates and culture media were subjected to SDS-PAGE (7.5% gel and 3 to 10% gel) and Western blot analysis. The viral S protein was detected by using an antipeptide antibody against the VHCR of the MHV-2 S protein, followed by a horseradish peroxidase-conjugated anti-rabbit IgG. GAPDH was employed as the loading control and was detected by using an anti-GAPDH antibody.

Fig 6

Inhibition of virus entry by treatment with protease inhibitors. (A) Effect of TMPRSS2 expression and exogenous trypsin treatment on virus entry into cells. MERS-CoV was adsorbed onto HeLa, HeLa-TMPRSS2, Vero, or Vero-TMPRSS2 cells for 1 h on ice, followed by the addition of trypsin (1 μg/ml). Cells were then incubated for a further 5 min at 37°C. The medium was changed, and the cells were incubated for an additional 5 h. (B) Effect of serine and cysteine protease inhibitors on virus entry. Vero or Vero-TMPRSS2 cells were infected with MERS-CoV in the presence of camostat (10 μM), EST (10 μM), or camostat plus EST and then incubated for a further 5 h at 37°C. (C) Effect of cathepsin inhibitors or endosome-tropic inhibitors on virus entry. Vero or Vero-TMPRSS2 cells were infected with MERS-CoV in the presence of bafilomycin A1 (100 nM) or inhibitors of cathepsin L (CatL; 10 μM), cathepsin B (CatB; 10 μM), cathepsin K (CatK; 10 μM), or cathepsin S (CatS; 10 μM). Cells were then incubated for a further 5 h at 37°C. Virus entry was quantified via real-time PCR by using an MERS-CoV-N probe set, as described in Table 1. Dimethyl sulfoxide (DMSO)-treated cells served as the negative control. Bars and error bars indicate the means and the standard deviations from six independent samples, respectively.

Fig 7

Characterization of human lung-derived cell lines based on the expression of cellular transcripts. (A) DPP4, TMPRSS2, HAT, cathepsin L, SP-D, and GAPDH (loading control) mRNA expression levels were measured in human lungs by using real-time PCR. (B) Total cellular RNA (0.1 μg) was isolated from WI-38, MRC-5, Calu-3, Vero, and HeLa cells and evaluated for the expression of DPP4, TMPRSS2, cathepsin L (CatL), SP-D, and GAPDH transcripts by using real-time PCR. Expression levels were compared to those in total RNA derived from human lung. ND, transcripts were not detected. (C) Cell lysates were subjected to SDS-PAGE (12.5% gel) and Western blot analysis. DPP4 was detected by using an anti-CD26 antibody, followed by a horseradish peroxidase-conjugated anti-rabbit IgG. GAPDH was employed as the loading control and was detected by using an anti-GAPDH antibody.

Fig 8

Effect of protease inhibitors on virus entry into human lung-derived cell lines. (A) WI-38, MRC-5, and Calu-3 cells were infected with MERS-CoV in the presence of camostat (Camo; 10 μM), EST (10 μM), camostat plus EST (Camo+EST), or leupeptin (10 μM). Cells were then incubated for a further 5 h at 37°C. (B) The concentration-dependent effects of serially diluted leupeptin or EST were tested in the three cell lines. Virus entry was quantified via real-time PCR by using an MERS-CoV-N probe set (Table 1). Dimethyl sulfoxide (DMSO)-treated cells served as the negative control. Bars and error bars indicate the means and the standard deviations from six independent samples, respectively.

Fig 9

Effect of protease inhibitors on HCoV-229E entry into WI-38 and MRC-5 cell lines. WI-38, MRC-5, and HeLa-TMPRSS2 cells were infected with HCoV-229E in the presence of camostat (Camo; 10 μM), EST (10 μM), camostat plus EST (Camo+EST), or leupeptin (100 μM). Cells were then incubated for a further 5 h at 37°C. Virus entry was quantified by real-time PCR by using an HCoV-229E-N probe set (Table 1). Dimethyl sulfoxide (DMSO)-treated cells served as the negative control. Bars and error bars indicate the means and the standard deviations from six independent experiments, respectively.

Fig 10

Inhibition of multistep MERS-CoV growth and virus-induced cell death by camostat. (A) Inhibition of multistep virus growth. Calu-3 cells in 24-well plates were infected with MERS-CoV (10 PFU) and incubated in the absence or presence of camostat (1, 10, or 100 μM). Viral RNA in the culture medium was isolated on the indicated days. (B) Comparison of virus growth among WI-38, MRC-5, and Calu-3 cells in the presence of camostat. Viral RNA in the culture medium was isolated at 3 days after MERS-CoV infection and quantified via real-time PCR by using an MERS-CoV-upE probe set (Table 1). DMSO-treated cells served as the negative control. Bars and error bars indicate the means and the standard deviations from six independent samples, respectively. (C) Inhibition of cytopathic effects by camostat. Calu-3, MRC-5, and WI38 cells were infected with MERS-CoV and treated with camostat as described above, incubated for 3 or 7 days, and observed by phase-contrast microscopy.