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. 2018 Sep 12;92(19):e00683-18.
doi: 10.1128/JVI.00683-18. Print 2018 Oct 1.

Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells

Shutoku Matsuyama  1 , Kazuya Shirato  2 , Miyuki Kawase  2 , Yutaka Terada  3 , Kengo Kawachi  3 , Shuetsu Fukushi  4 , Wataru Kamitani  3
Affiliations

Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells

Shutoku Matsuyama et al. J Virol. .

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. In this study, we reexamined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK inhibited not only the enzymatic activity of furin but also those of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin.IMPORTANCE Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be reexamined carefully. Here we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.

Keywords: TMPRSS2; cathepsin L; coronavirus; dec-RVKR-CMK; furin.

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Figures

FIG 1
FIG 1
Comparison of three assays to quantify virus cell entry. (A to C) Cell entry by pseudotyped or authentic MERS-CoV. Vero/TMPRSS2 cells in 96-well plates were infected with the serially diluted viruses indicated at the top. The relationship between inoculated virus titer (x axis) and data values (average of two experiments) for each assay (y axis) is shown. (A) GFP-positive cells were counted at 20 h postinfection. (B) Luciferase activity in cells was measured at 20 h postinfection. (C) The amount of viral mRNA in cells at 6 h postinfection was measured by real-time PCR. (D to F) Effect of a furin inhibitor on cell entry by pseudotyped or authentic MERS-CoV. Vero/TMPRSS2 cells were inoculated with viruses in the presence or absence of the furin inhibitor dec-RVKR-CMK. Virus entry was measured using appropriate assays. (D) Effect of dec-RVKR-CMK on cell entry by VSV-ΔG/GFP-MERS-S. Cells were inoculated with 103 infectious units of VSV-ΔG/GFP-MERS-S (MOI = 0.01). GFP-positive cells were counted at 20 h postinfection (n = 4). (E) Effect of dec-RVKR-CMK on cell entry by VSV-ΔG/Luc-MERS-S. Cells were inoculated with 104 infectious units of VSV-ΔG/Luc-MERS-S (MOI = 0.1). Luciferase activity in cells was measured at 20 h postinfection (n = 4). Data are presented on linear (left) and logarithmic (right) scales. (F) Effect of dec-RVKR-CMK on cell entry by authentic MERS-CoV. Cells were inoculated with 105 infectious units of authentic MERS-CoV (MOI = 1). The amount of viral mRNA in cells at 6 h postinfection was measured by real-time PCR (n = 4). A two-tailed Student t test was used to analyze statistical significance.
FIG 2
FIG 2
Effect of a furin inhibitor on MERS-CoV entry into Calu-3 cells. Calu-3 human bronchial epithelial cells were pretreated for 30 min with increasing concentrations (0 to 100 μM) of the furin inhibitor dec-RVKR-CMK. E64d (10 μM), camostat (10 μM), or a combination of both was used as a comparison control. The cells were then infected with 105 PFU of MERS-CoV (MOI = 1) in the presence of inhibitor. The amount of viral mRNA in Calu-3 cells at 6 h postinfection was measured by real-time PCR (n = 4). A two-tailed Student t test was used to analyze statistical significance.
FIG 3
FIG 3
Comparison of transcripts in LoVo cells. (A) Expression of mRNA in Calu-3, LoVo, and Huh-7 cells. Total cellular RNA (0.1 μg) was evaluated for expression of GAPDH, DPP4, furin, cathepsin L, TMPRSS2, and HAT transcripts using real-time PCR (n = 4). ND, no transcripts were detected. (B) Electropherograms of furin cDNA. The mRNAs isolated from Calu-3 and LoVo cells were reverse transcribed and amplified using a thermal cycler and used for DNA sequencing. (C) Viral yield in LoVo cells. Confluent Calu-3 cells and LoVo cells were grown in 96-well plates and infected with MERS-CoV at an MOI of 0.001 for 24 h. Cell-free supernatants were harvested, and infectious viral titers were measured in a standard plaque assay using Vero/TMPRSS2 cells (n = 4). A two-tailed Student t test was used to analyze statistical significance.
FIG 4
FIG 4
Effect of furin inhibitor on MERS-CoV and SARS-CoV entry into LoVo cells. (A) Effect of inhibitors on MERS-CoV entry. LoVo cells were pretreated for 30 min with the furin inhibitor dec-RVKR-CMK (concentration, 0 to 100 μM). E64d (10 μM), camostat (10 μM), or a combination of both was used as a comparison control. The cells were then infected with 105 PFU of MERS-CoV (MOI = 1) in the presence of inhibitor. The amount of viral mRNA in LoVo cells at 6 h postinfection was measured by real-time PCR (n = 4). (B) Effects of inhibitors on SARS-CoV entry. SARS-CoV was used instead of MERS-CoV; all experiments were carried out as described for panel A. A two-tailed Student t test was used to analyze statistical significance.
FIG 5
FIG 5
Effect of mutations in the furin cleavage site of the MERS-CoV S protein. (A) Cleavage of the S protein on virions. Wild-type (wt) and mutant MERS-CoV lacking furin cleavage sites within the S protein (generated in Vero/TMPRSS2 cells) was subjected to Western blot analysis with an anti-S polyclonal antibody. (B) Cleavage of S protein in cells. The wt and mutant viruses shown in panel A were used to infect Vero/TMPRSS2, Huh-7, and LoVo cells, and cell lysates prepared at 20 h postinfection were subjected to Western blot analysis. (C) Entry of mutant viruses into LoVo and Huh-7 cells. The cells were infected with 104 PFU of MERS-CoV (MOI = 0.1). The amount of viral mRNA in cells at 6 h postinfection was measured by real-time PCR (n = 4). (D) Effect of mutations on virus entry. LoVo cells were pretreated for 30 min with the furin inhibitor dec-RVKR-CMK (concentration of 100 μM). Then cells were infected with 104 PFU of MERS-CoV (MOI = 0.1) in the presence of inhibitor. The amount of viral mRNA in cells at 6 h postinfection was measured by real-time PCR (n = 4). Data are expressed as the fold change in viral mRNA levels relative to that in the absence of dec-RVKR-CMK. A two-tailed Student t test was used to analyze statistical significance.
FIG 6
FIG 6
S protein activation by exogenous furin. (A) Activity of commercial furin. The enzymatic activity of recombinant human furin used in the experiment was confirmed using the fluorescent substrate BOC-RVRR-AMC. (B) Exogenous furin does not trigger cell entry by virus. LoVo cells in 96-well plates were inoculated with MERS-CoV (MOI of 0.1) for 30 min and then treated with furin (2,000 units/ml) or trypsin (10 μg/ml) at 37°C for 10 min. Cells were then cultured at 37°C for 6 h. The amount of viral mRNA was measured by real-time PCR (n = 4). A two-tailed Student t test was used to analyze statistical significance. (C) Exogenous furin does not induce syncytium formation. LoVo cells infected with MERS-CoV (MOI of 0.01) were cultured for 20 h and then treated with furin (2,000 units/ml) or trypsin (10 μg/ml) for 5 h. (D) Cleavage of S protein by exogenous furin. MERS-CoV propagated in Vero cells was treated with the indicated concentrations of trypsin or furin at 37°C for 60 min. Western blot analysis was performed using an anti-S polyclonal antibody.
FIG 7
FIG 7
Timing of inhibitor addition to block entry of MERS-CoV into cells. (A) Dec-RVKR-CMK (100 μM), camostat (10 μM) plus E64d (10 μM), or lopinavir (30 μM) was added to LoVo cells (for 30 min on ice) at the indicated times before and after MERS-CoV inoculation. The amount of viral mRNA in cells at 6 h postinfection was measured by real-time PCR (n = 1). (B) Calu-3 cells were used instead of LoVo cells; all experiments were carried out as described for panel A (excluding lopinavir treatment) (n = 1).
FIG 8
FIG 8
Effect of furin inhibitor on commercial proteases and cell surface TMPRSS2. (A) Inhibitory effect of dec-RVKR-CMK on commercial proteases. Degradation products of fluorescein-labeled casein generated by treatment with commercial proteases, cathepsin L (20 μg/ml), cathepsin B (20 μg/ml), trypsin (50 μg/ml), papain (0.3 units/ml), proteinase K (0.3 units/ml), dispase (3 units/ml), elastase (200 μg/ml), and chymotrypsin (10 μg/ml), at 37°C for 30 min in the presence of dec-RVKR-CMK (serially diluted 10-fold) were quantified by fluorometry (n = 3). Data are expressed as average percentages relative to that in the absence of dec-RVKR-CMK. N, no inhibitor treatment. (B) E64d and bafilomycin A1 do not inhibit commercial proteases. E64d or bafilomycin A1 was used instead of dec-RVKR-CMK; all experiments were carried out as described for panel A (proteases tested were trypsin, chymotrypsin, and elastase). (C) Inhibitory effect of dec-RVKR-CMK against TMPRSS2, as assessed in the FFWO assay. A high titer of MERS-CoV (MOI = 10) was adsorbed onto Vero/TMPRSS2 or Vero cells (on ice for 1 h). Cells were then incubated at 37°C in the presence of the inhibitor. After 5 h, cells were fixed and stained with crystal violet. DMSO, dimethyl sulfoxide.
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