2018 Middle East respiratory syndrome coronavirus spike protein is not activated directly by cellular furin during viral

1

Middle East respiratory syndrome coronavirus spike protein is not activated 1

directly by cellular furin during viral entry into target cells 2

3

Shutoku Matsuyama,a#

Kazuya Shirato,a Miyuki Kawase,

a Yutaka Terada,

c Kengo 4

Kawachi,c Shuetsu Fukushi,

b and Wataru Kamitani

c 5

6

aDepartment of Virology III, National Institute of Infectious Diseases, Tokyo, Japan 7

bDepartment of Virology I, National Institute of Infectious Diseases, Tokyo, Japan 8

cLaboratory of Clinical Research on Infectious Diseases, Osaka University, Osaka, Japan 9

10

Running Head: Cell entry by MERS-CoV is not dependent on furin 11

12

#Address correspondence to Shutoku Matsuyama, [email protected]. 13

14

JVI Accepted Manuscript Posted Online 18 July 2018J. Virol. doi:10.1128/JVI.00683-18Copyright © 2018 American Society for Microbiology. All Rights Reserved.

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ABSTRACT 15

Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular 16

proteases to enter cells. A previous report shows that furin, which is distributed mainly in 17

the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically 18

activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion 19

between the viral and cellular membranes. Here, we re-examined furin usage by 20

MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular 21

proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of 22

MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry 23

into furin-deficient LoVo cells. In addition, dec-RVKR-CMK not only inhibited the 24

enzymatic activity of furin but also that of cathepsin L, cathepsin B, trypsin, papain, and 25

TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no 26

evidence that the S protein was activated by exogenous furin. Therefore, we conclude that 27

furin does not play a role in entry of MERS-CoV into cells, and that the inhibitory effect 28

of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin. 29

30

IMPORTANCE 31

Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV 32

utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. 33

However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. 34

Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest 35

that previous studies in the virology field based on dec-RVKR-CMK should be 36

re-examined carefully. Here, we describe appropriate experiments that can be used to 37

assess the effect of protease inhibitors on virus cell entry. 38

39

40


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INTRODUCTION 41

Many species of enveloped virus utilize host cellular proteases to infect cells (1). Two 42

major mechanisms are responsible for proteolytic activation of viral spike (S) 43

glycoproteins. In the case of human immunodeficiency virus and highly pathogenic avian 44

influenza viruses, a cellular protease called furin cleaves the viral glycoprotein during 45

biogenesis, thereby converting the precursor glycoprotein to its fusion-competent state 46

(1–3). Alternatively, the S protein of viruses such as severe acute respiratory syndrome 47

coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus 48

(MERS-CoV) is cleaved by cell surface or endosomal proteases such as transmembrane 49

protease serine 2 (TMPRSS2), HAT, furin, trypsin, elastase, or cathepsin L. This cleavage 50

triggers conformational changes during viral entry after the receptor-binding step (2–14). 51

In the presence of extracellular or cell surface proteases such as elastase or TMPRSS2, 52

MERS-CoV enters cells after binding to the cell surface receptor; however, in their 53

absence, MERS-CoV utilizes cathepsin L in the late endosome (10). Cleavage releases 54

the receptor-binding S1 subunit from the membrane fusion S2 subunit, which triggers 55

conformational changes in the S2 subunit to induce membrane fusion (2). 56

The role of furin in activating the MERS-CoV S protein is controversial. Furin is a 57

proprotein convertase responsible for maturation of a huge number of inactive proteins; it is 58

localized principally in the trans-Golgi network, from where it is cycled to the cell 59

surface and the endosomes, which are organelles used by MERS-CoV for cell entry (11). 60

Of the furin inhibitors reported previously (15–17), dec-RVKR-CMK and a proprotein 61

convertase inhibitor (PCI) are often used in virology experiments. Gierer et al. used a PCI 62

to show that enzymatic processing during biogenesis of the MERS-CoV S protein by 63

proprotein convertases such as furin is not required for infectivity; however, they stated 64

that the host cell protease is required for S protein activation during viral uptake into 65

target cells (14). By contrast, Park et al. used dec-RVKR-CMK to show that mutated 66


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MERS-CoV (in which the furin cleavage site was masked) tended to enter cells via the 67

endosomal pathway, while the wild-type (wt) virus entered at the cell via cell surface 68

proteases; this indicates that cleavage of the S protein during biogenesis determines the 69

tropism of the virus for different cell types (8). Furthermore, Millet and Whittaker 70

reported that the MERS-CoV S protein harbors furin cleavage sequences at the S1/S2 and 71

S2’ sites; these sites could be targeted by host cellular furin during cell entry. Indeed, 72

non-cytotoxic concentrations (2.5–100 μM) of dec-RVKR-CMK prevented entry of 73

pseudotyped and authentic MERS-CoV (13). Burkard et al. used dec-RVKR-CMK to 74

show that murine coronavirus mouse hepatitis virus (MHV) also uses furin for viral cell 75

entry (12). However, furin is thought to make only a minor contribution to viral cell entry 76

because MERS-CoV entry into some cell lines (even furin-expressing cells) is almost 77

completely blocked by simultaneous treatment with a TMPRSS2 inhibitor (camostat 78

mesylate) and a cathepsin inhibitor (E64d) (10). Here, we re-examined the role of furin 79

during MERS-CoV infection to clarify when and how it is involved in virus cell entry. 80

The findings question our current understanding of host protease usage underlying 81

MERS-CoV infection. 82

83

RESULTS 84

Evaluation of an appropriate assay to monitor virus cell entry 85

First, we examined cell entry by three types of virus: 1) a pseudotyped vesicular 86

stomatitis virus (VSV) bearing a MERS-CoV S protein in which the VSV-G gene was 87

replaced by the green fluorescent protein (GFP) gene (VSV-ΔG/GFP-MERS-S); 2) the 88

same virus in which the GFP gene was replaced by the luciferase (Luc) gene 89

(VSV-ΔG/Luc-MERS-S); and 3) authentic MERS-CoV. These viruses were used to infect 90

Vero/TMPRSS2 cells (18), which are highly susceptible to MERS-CoV (10). Entry of 91

these viruses into cells was measured using appropriate assays (see Materials and 92

methods). The range for VSV-ΔG/GFP-MERS-S was narrow (1–3 log10 GFP-cell 93


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counts) (Fig. 1A), whereas that for luciferase was broad (1–5 log10 Luc unit) (Fig. 1B); 94

however, the range for authentic MERS-CoV (assessed by real-time PCR) was broader 95

still (2–7 log10 copies of viral mRNA) (Fig. 1C). 96

To evaluate the suitability of the assays for the furin inhibition experiments, we examined 97

the effect of a furin inhibitor (dec-RVKR-CMK; 100 μM) on viral entry into cells. 98

Infection with VSV-ΔG/GFP-MERS-S in the presence of dec-RVKR-CMK led to a fall in 99

the number of GFP-positive cells by 60% (0.38 log) (Fig. 1D). Infection by 100

VSV-ΔG/Luc-MERS-S in the presence of the inhibitor led to a fall of 40% (0.21 log) (Fig. 101

1E, left panel). By contrast, infection by authentic MERS-CoV led to a 97% (1.53 log) 102

reduction in the viral mRNA copy number (Fig. 1F). The pseudotyped virus encoding 103

GFP is suitable for measuring the percentage value of infection whithin a narrow range. 104

However, we surmised that VSV-ΔG/Luc-MERS-S would not provide reliable results 105

because the 40% reduction observed in the assay is negligible on a logarithmic scale (Fig. 106

1E, right panel), we must consider the quantifiable range for this virus was 4 logs, and the 107

luminometer used in the assay has a dynamic range of 9 logs. Therefore, we used 108

authentic MERS-CoV for experiments designed to measure the inhibitory effects of 109

dec-RVKR-CMK. 110

111

Entry of MERS-CoV into Calu-3 cells in the presence of a furin inhibitor 112

To examine the effect of cellular furin on MERS-CoV infection, we infected Calu-3 cells 113

(which are derived from human bronchial epithelial cells) with the authentic MERS-CoV 114

in the presence of inhibitors. After a 6 h incubation at 37°C, cellular RNA was isolated 115

and real-time PCR was carried out to quantify the amount of viral mRNA. As shown in 116

Figure 2, a TMPRSS2 inhibitor (camostat mesylate; 10 μM) suppressed virus entry by 117

100-fold; similar results were observed after treatment with 20 μM dec-RVKR-CMK. 118

E64d had little effect on virus entry, suggesting that MERS-CoV mainly uses the 119


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TMPRSS2/cell surface pathway rather than the cathepsin/endosome pathway to enter 120

Calu-3 cells. Taken together, the results suggest that either furin is essential for S protein 121

activation or that dec-RVKR-CMK suppresses both furin and TMPRSS2. 122

123

Susceptibility of furin-deficient LoVo cells to infection by MERS-CoV 124

LoVo cells (derived from human colon adenocarcinoma cells) lack furin activity because 125

they harbor two distinct mutant alleles of the furin gene (1286T and 1639C) (19, 20). We 126

purchased LoVo cells from the American Type Culture Collection (ATCC) and quantified 127

expression of mRNA encoding proteins involved in cell entry by MERS-CoV. We 128

confirmed that, like Calu-3 and Huh-7 (derived from human liver carcinoma) cells, LoVo 129

cells expressed DPP4, furin, cathepsin L, and TMPRSS2; however, unlike Calu-3 and 130

Huh-7 cells, LoVo cells expressed HAT (Fig. 3A). Furin expression was highest in Huh-7 131

cells. We also confirmed deletion and substitution (1286T and 1639C) mutations within 132

the furin mRNA sequence (Fig. 3B) (19, 20). Next, we inoculated MERS-CoV onto LoVo 133

cells and measured virus propagation at 24 h. As reported previously (21), MERS-CoV 134

infected and replicated in LoVo and Calu-3 cells (Fig. 3C). This indicates that furin is not 135

essential for MERS-CoV infection. 136

137

Entry of MERS-CoV into furin-deficient LoVo cells 138

Next, we examined the effect of protease inhibitors on MERS-CoV entry into LoVo cells. 139

E64d suppressed entry of MERS-CoV, but camostat did not, suggesting that the 140

cathepsin/endosomal pathway rather than the TMPRSS2/cell surface pathway is 141

dominant in LoVo cells (Fig. 4A). Treatment with 100 μM dec-RVKR-CMK completely 142

suppressed entry of MERS-CoV, similar to co-treatment with camostat and E64d (Fig. 143

4A). Furthermore, cell entry by SARS-CoV, in which the S protein lacks furin cleavage 144

sites (13), was also suppressed by dec-RVKR-CMK (Fig. 4B). 145


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MERS-CoV mutants in which the S protein lacks furin cleavage sites at R748 (the S1/S2 146

site) and/or R884 (the S2’ site) were generated using a recently developed reverse 147

genetics system (22). Western blot analysis of S proteins harboring mutations at R748 148

(R748S or R748S/R884S) detected no 80 kD cleavage product on virions (Fig. 5A, lanes 149

2 and 4). To characterize the cleavability of these S proteins within cells, Vero/TMPRSS2, 150

Huh-7 (expressing high levels of furin), and LoVo (lacking furin) cells were infected with 151

viruses and cell lysates were examined by western blotting. The S proteins of wt and 152

R884 mutant viruses in Vero/TMPRSS2 and Huh-7 cells were cleaved, but those of R748 153

mutant viruses (R748S or R748S/R884S) were not (Fig. 5B). By contrast, none of the S 154

proteins were cleaved in LoVo cells (Fig. 5B). This confirmed that LoVo cells lack furin 155

activity and that the R748 mutation in S protein lies within the furin cleavage site. To 156

assess the furin-dependent cell entry of these mutant viruses, they were inoculated onto 157

Huh-7 or LoVo cells and the amount of viral mRNA at 6 h post-infection was measured 158

by real-time PCR. No significant difference was observed between wt and mutant viruses 159

in Huh-7 cells (Fig. 5C), indicating that high levels of furin expressed by these cells did 160

not affect S protein activation during viral entry. Treatment with dec-RVKR-CMK 161

suppressed cell entry by both wt MERS-CoV and mutant MERS-CoV in which the S 162

protein lacks furin cleavage sites (Fig. 5D). These results indicate that entry of 163

furin-deficient cells by virus lacking furin cleavage sites is blocked by the furin inhibitor, 164

suggesting that the molecule targeted by dec-RVKR-CMK during MERS-CoV entry is 165

not furin. 166

167

Exogenous furin does not activate the S protein 168

Next, we examined direct activation of the S protein by exogenous furin. First, we 169

confirmed that commercial furin had the advertised level of activity by testing it using a 170

furin substrate, BOC-RVRR-AMC (Fig. 6A). Treatment with exogenous trypsin, but not 171


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exogenous furin, increased virus entry into LoVo cells (Fig. 6B) and cell-cell fusion by 172

MERS-CoV-infected LoVo cells (Fig. 6C). Therefore, we concluded that furin does not 173

activate the MERS-CoV S protein. In addition, western blot analysis detected very small 174

amounts of cleaved S protein (80 kD) in non-protease-treated viruses propagated in Vero 175

cells (Fig. 6D, lane 1); similar results were obtained for viruses propagated in 176

Vero/TMPRSS2 cells (Fig. 5A). No 80 kD S protein was detected when cells were treated 177

with exogenous furin (2000 units/ml), although the 150 kD and 50 kD products were 178

detected (Fig. 6D, lane 5). Of note, the S protein-harboring virions used for the 179

experiments in Figure 6D were propagated in Vero cells cultured in medium lacking 180

trypsin (a non-enzymatic cell dissociation solution (C5914; Sigma) was used for cell 181

passage). The intensity of the 80 kD band increased only when cells were exposed to 182

exogenous trypsin (0.1 μg/ml) (Fig. 6D, lanes 3 and 7). Therefore, we conclude that the S 183

protein is cleaved by furin during biogenesis, not on virions after exit the cells. 184

185

The inhibitor dec-RVKR-CMK targets the early stage of MERS-CoV infection 186

Our next task was to identify the true target of dec-RVKR-CMK during MERS-CoV 187

infection. First, we examined the effect of dec-RVKR-CMK on endocytosis. pHrodo™ 188

dextran, which is non-fluorescent at neutral pH but exhibits increased fluorescence as the 189

pH becomes acidic, was used as a tracker of endocytic internalization and lysosomal 190

sequestration within live cells. In Calu-3 cells, bafilomycin A1, which blocks 191

acidification of endosomes, clearly inhibited development of pHrodo red, but 192

dec-RVKR-CMK, camostat, and E64d did not (data not shown); this suggests that 193

dec-RVKR-CMK has no effect on endocytosis. 194

Next, to clarify the time point at which inhibitors block MERS-CoV infection, LoVo cells 195

were treated with 100 μM dec-RVKR-CMK or a mixture of 10 μM E64d and 10 μM 196

camostat (E64d/camostat) during infection with MERS-CoV. Lopinavir, which targets 197


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MERS-CoV 3CL protease and blocks viral RNA replication, was used for comparison. 198

The inhibitors were added at the indicated time points, and cellular RNA was isolated 6 h 199

after infection. Samples collected at 6 h post-infection were used as a control for no 200

inhibitor treatment. The amount of viral mRNA was quantified by real-time PCR. 201

Dec-RVKR-CMK and E64d/camostat showed an inhibitory effect within 30 min after 202

infection (Fig. 7A). By comparison, addition of lopinavir at 2 h post-infection still 203

inhibited the viral RNA replication (Fig. 7A). In Calu-3 cells, dec-RVKR-CMK and 204

E64d/camostat showed a similar effect; both of these inhibitors act at the very early stage 205

of infection (within 30 min) (Fig. 7B). Taken together, the data indicate that 206

dec-RVKR-CMK targets an early step during MERS-CoV entry: potentially S protein 207

activation by TMPRSS2 and cathepsin L. 208

209

Inhibitory effect of dec-RVKR-CMK on commercial proteases and TMPRSS2 210

Next, we examined the inhibitory effect of dec-RVKR-CMK on various proteases. We 211

purchased proteases from commercial sources and tested them using a fluorescent 212

protease assay kit, which measures degradation products of fluorescein-labeled casein. 213

Unfortunately, the kit was unable to detect furin activity because casein does not contain 214

the furin cleavage site. First, 10-fold serially diluted protease was incubated with the 215

substrate to identify the appropriate concentration representing the linear phase of the 216

reaction at 30 min; this was used for the experiments described below. Next, appropriate 217

concentrations of various proteases required to degrade the substrate were mixed with 218

dec-RVKR-CMK and inhibition kinetics were measured. Figure 8A shows that a high 219

concentration (100 μM) of dec-RVKR-CMK completely suppressed the activity of 220

cathepsin L, cathepsin B, trypsin, and papain, partially suppressed that of proteinase K 221

and dispase, and slightly suppressed that of elastase and chymotrypsin. Neither E64d nor 222

bafilomycin A1 (used as a control) suppressed the activity of trypsin, chymotrypsin, or 223


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elastase (Fig. 8B). 224

To examine the inhibitory effect of dec-RVKR-CMK on TMPRSS2, we performed a 225

fusion-from-without (FFWO) assay as described previously (10). This assay detects viral 226

S protein-mediated cell-cell fusion activated by TMPRSS2 on the cell surface; the assay 227

excludes the effects of inhibitors on virus replication, meaning that it detects TMPRSS2 228

activity directly. Briefly, a high titer (MOI = 10) of MERS-CoV was adsorbed onto 229

Vero/TMPRSS2 cells on ice for 1 h. The cells were then shifted to 37°C in the presence 230

or absence of inhibitors. Cell-cell fusion was first observed at 3 h after warming. The 231

dec-RVKR-CMK inhibitor (100 μM) suppressed cell-cell fusion completely, as did 232

camostat (10 μM; used as the control) (Fig. 8C). These results strongly suggest that 233

dec-RVKR-CMK targets cell surface TMPRSS2, which plays a role in cell entry by 234

MERS-CoV. 235

236

DISCUSSION 237

Cellular furin plays a role in virus infection and numerous other biological phenomena, 238

most of which were identified by experimental observations using the furin inhibitor 239

dec-RVKR-CMK (11, 13, 23–25). However, the results presented herein indicate that 240

dec-RVKR-CMK inhibited not only furin but also cathepsin L and TMPRSS2 (Fig. 8A 241

and C). In addition, we observed that dec-RVKR-CMK blocked entry of viruses 242

harboring an S protein lacking furin cleavage sites; it even blocked entry into 243

furin-deficient LoVo cells (Fig. 5D). This suggests that the results of previous studies 244

using dec-RVKR-CMK may have to be re-examined. 245

In addition, virus entry or cell-cell fusion assays provided no evidence that the S protein 246

was activated by exogenous furin (Fig. 6B and C). A previous study claims to show direct 247

evidence of S protein activation by furin because induction of cell-cell fusion in S 248

protein-expressing Huh-7 cells was observed after removing dec-RVKR-CMK from the 249


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culture medium and adding exogenous furin (13). However, exogenous furin may not be 250

required to induce cell-cell fusion of Huh-7 cells because these cells induce cell-cell 251

fusion after MERS-CoV infection in the absence of proteases (26). 252

Here, we discuss the use of host proteases by MERS-CoV for cell entry. Studies show 253

clearly that TMPRSS2 activates the MERS-CoV S protein (4, 10, 27). Expression of 254

TMPRSS2 at the cell surface induces both virus entry into cells and cell-cell fusion of S 255

protein-expressing cells (4, 10, 27). Furthermore, these phenomena are suppressed by the 256

serine protease inhibitors camostat mesylate and nafamostat mesylate (4, 10, 28). In 257

addition, other extracellular proteases such as trypsin and elastase activate the 258

MERS-CoV S protein in a manner similar to TMPRSS2 (10). 259

However, cathepsin L usage by MERS-CoV remains controversial because there is no 260

clear evidence that the S protein is activated by exposure to exogenous cathepsin L. A 261

previous study shows that cell-cell fusion induced by exogenous cathepsin L in 262

SARS-CoV S protein-expressing cells is insufficient (29). Also, our previous study shows 263

that cathepsin L requires chlorpromazine, a membrane-permeable cationic drug that 264

lowers the energy requirement for membrane fusion, to induce a small increase in cell 265

entry by murine coronavirus (30). Therefore it is unclear whether cathepsin L plays a role 266

in activating the coronavirus S protein. However, it is at least certain that the enzymatic 267

activity of cathepsin is necessary for proteolytic activation of the MERS-CoV cell entry 268

because expression of TMPRSS2 overcomes blockade of viral cell entry by cathepsin 269

inhibitors (10). 270

Taken together, the results presented herein do not support a role for cellular furin during 271

direct activation of MERS-CoV S protein for viral cell entry. These findings are 272

compatible with those of a previous study by Gierer et al. showing that MERS-CoV does 273

not require furin for infectivity (14). Park et al. report that the only role played by furin 274

during MERS-CoV infection is to determine the cell tropism of the virus (8). What is 275


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certain that the MERS-CoV S protein pre-cleaved by furin at the S1/S2 site still requires 276

cell surface/extracellular and endosomal proteases such as TMPRSS2, elastase, and 277

cathepsin L, for cleavage at the S2’ site. 278

279

280

MATERIALS AND METHODS 281

Cells and viruses 282

LoVo and Calu-3 cell lines were cultured as recommended by the ATCC. Vero cells 283

obtained from the ATCC and Vero cells expressing TMPRSS2 (Vero/TMPRSS2) (18) 284

were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich, USA) 285

supplemented with 5% heat-inactivated fetal bovine serum (Gibco-BRL, USA). 286

MERS-CoV EMC strain and SARS-CoV Frankfurt 1 strain were propagated in Vero 287

cells. 288

289

Generation of a pseudotyped virus 290

The VSV-pseudotyped virus expressing GFP or luciferase and harboring the MERS-CoV 291

S protein was prepared as previously described (31). Briefly, at 24 h post-transfection 292

with pKS-MERS-St16, 293T cells were infected with VSVΔG-G/GFP or VSVΔG-G/Luc 293

at a MOI of 0.1. After absorption for 1 h, the inoculum was replaced with culture medium. 294

After a further incubation for 24 h, the culture supernatants were collected and stored at 295

-80°C. The titer of VSV-pseudotyped viruses was determined in Vero cells. For the 296

VSV-pseudotyped virus expressing GFP, images were captured under a BZ8000 297

microscope (Keyence, Japan) and GFP-positive cells were counted using image 298

measurement and analysis software (VH-H1A5 version 2.6; Keyence). For the 299

VSV-pseudotyped virus expressing luciferase, cells were lysed and assayed for luciferase 300

activity using a luciferase assay kit (Promega, USA) and a Glomax 20/20 luminometer 301


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instrument (Promega). 302

303

Generation of recombinant MERS-CoV from bacterial artificial chromosome (BAC) 304

plasmids 305

A bacterial artificial chromosome (BAC) clone carrying the full-length infectious genome 306

of the MERS-CoV EMC2012 strain (termed pBAC-MERS-wt) was used to generate 307

recombinant MERS-CoV (22). Amino acid substitutions at the P1 site within the fusion 308

cleavage sites in the S protein were generated by modification of the pBAC-MERS-wt 309

(the template) using a Red/ET Recombination System Counter-Selection BAC 310

Modification Kit (Gene Bridges, Heidelberg, Germany). This yielded 311

pBAC-MERS-S/R748S, pBAC-MERS-S/R884S, and pBAC-MERS-S/R748S/R884S. 312

Huh-7 cells were grown to approximately 60% confluence in a 6-well plate (VIOLAMO, 313

Japan) and then transfected with 4 µg of the indicated BAC DNA using the 314

X-tremeGENE 9 DNA Transfection Reagent (Roche). Transfected cells were cultured at 315

37°C for the indicated times, and the culture supernatants and cell pellets were collected. 316

The culture supernatants were inoculated onto a 10 cm dish (VIOLAMO) containing 317

Huh-7 cells and cultured overnight. Infected cells were incubated at 37°C for 3 or 4 days 318

until a cytopathic effect (CPE) was observed. Culture supernatants were collected, 319

centrifuged at 2500×g for 5 min at 4°C, and stored at -80°C. 320

321

Proteases and protease inhibitors 322

The following proteases were used: cathepsin L (16-12-030112; Athens, USA), cathepsin 323

B (16-12-030102; Athens), trypsin (T8802; Sigma, USA), papain (53J6521; Worthington, 324

USA), proteinase K (166-21051; Wako, Japan), dispase (1 276 921; Roche, Switzerland), 325

elastase (Sigma; E-0258), chymotrypsin (Sigma; C-3142), and furin (P8077; NEB, UK). 326

The following inhibitors were used: dec-RVKR-CMK (3501; Tocris, UK), E64d (330005; 327


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Calbiochem, USA), camostat (3193; Tocris Bioscience, UK), bafilomycin A1 (B1793; 328

Sigma), and lopinavir (SML1222; Sigma). 329

330

Cell entry assay for authentic MERS-CoV 331

Confluent cells in 96-well plates were pretreated for 30 min with inhibitors. Cells were 332

then inoculated with MERS-CoV and incubated (with the inhibitors) for 1 h on ice, 333

followed by culture at 37°C for 6 h. Cellular RNA was isolated by addition of Isogen 334

reagent (315-02504; Nippon Gene, Japan). A real-time PCR assay was performed to 335

quantify the amount of newly synthesized subgenomic MERS-CoV RNA using the 336

primers and probes described previously (10). PCR analysis was performed in a 337

LightCycler-Nano instrument (Roche Diagnostics, Switzerland). 338

339

Quantification of transcripts in LoVo cells 340

Total RNA was isolated from LoVo cells using the Isogen reagent. Real-time PCR was 341

performed to quantify expression of mRNA encoding GAPDH, DPP4, furin, cathepsin L, 342

TMPRSS2, and HAT using the primers and probes described previously (10). 343

Comparative expression of mRNA was calculated from a calibration line obtained by 344

stepwise dilution (10-fold) of RNA samples. 345

346

Quantification of furin activity 347

Recombinant human furin (P8077; New England Biolabs, UK) was mixed with 100 μM 348

furin substrate (boc-RVRR-AMC; I-1645; Bachem, Switzerland) in phosphate buffered 349

saline containing 1 mM CaCl2 (32). After 30 min at 37°C, fluorescence analysis was 350

performed using a Power Scan HT instrument (DS Pharma, Japan) fitted with fluorescein 351

excitation/emission filters (360/480 nm). 352

353


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354

Western blot analysis 355

To detect cleaved S protein in virions, viral supernatant was mixed with recombinant 356

human furin (P8077; New England Biolabs) or trypsin (T-8802; Sigma) in furin reaction 357

buffer (20 mM HEPES, pH 7.5, 0.1% Triton X-100, 0.2 mM CaCl2, and 0.2 mM 358

β-mercaptoethanol) and incubated at 37°C for 1 h. One quarter volume of sample buffer 359

(30% glycerol, 250 mM Tris pH 6.8, 2.5% SDS, a small amount of Bromophenol blue, 360

100 mM DTT, and 1 mM pefablock SC) was added to the reaction and boiled for 5 min. 361

Samples were loaded onto SDS-PAGE (3–10% gradient) gels (e-PAGEL; ATTO, Japan), 362

transferred to a PVDF membrane (Immobilon-P; Millipore, USA), and soaked in 363

ImmunoBlock (CTKN001; DS Pharma Biomedical, Japan). Western blot analysis was 364

carried out using an anti-S2 antibody and anti-rabbit IgG (sc-2054; Santa Cruz Biotech, 365

USA). Immunoreactive bands were visualized with an enhanced chemiluminescence kit 366

(ECL, RPN2232; GE Healthcare, USA) and a LAS-3000 instrument (Fuji, Japan). 367

To detect the S protein in MERS-CoV-infected cells, cells were dissolved in sample 368

buffer and subjected to western blot analysis as described above. After detection by an 369

anti-S2 antibody, the membrane was soaked in stripping buffer (46428; ThermoFisher, 370

USA) for 5 min at room temperature to remove the antibodies and then rinsed 10 times 371

with rinse buffer (20845; Millipore). The membrane was then blocked and re-probed with 372

an anti-GAPDH antibody (IMG-5143A; IMGENEX, USA), followed by anti-rabbit IgG. 373

374

Fluorescent protease assay for commercial proteases 375

The activity of the proteases listed above was quantified using a fluorescent protease 376

assay kit (23266; Pierce, USA), which measures the degradation products of 377

fluorescein-labeled casein. To measure the activity of neutral pH-dependent proteases 378

(trypsin, papain, proteinase K, dispase, elastase, and chymotrypsin), experiments were 379


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carried out according to the manufacturer’s protocol. To measure the activity of low 380

pH-dependent cathepsin L and cathepsin B, the substrate (which has neutral 381

pH-dependent fluorescence) was first dissolved in low pH buffer (50 mM sodium acetate 382

pH 5.0, 1 mM EDTA, and 5 mM DTT). After a 30 min incubation with cathepsin at 37°C, 383

1/20 volume of 1 M Tris-HCl buffer pH 8.0 was added to render the fluorescein 384

detectable at neutral pH. Fluorescence analysis was performed using a Power Scan HT 385

instrument (DS Pharma, Japan) fitted with fluorescein excitation/emission filters 386

(485/528 nm). 387

388

FFWO assay 389

The FFWO assay was performed as previously described (10). Briefly, Vero/TMPRSS2 390

cells (105 cells) in 96-well plates were inoculated with a high titer (10

6 PFU) of authentic 391

MERS-CoV. Cells were then incubated at 37°C in the presence of inhibitors. After 392

incubation for 5 h, cells were fixed with 4% formaldehyde and stained with crystal violet. 393

394

Statistical analysis 395

Statistical significance was assessed using a two-tailed Student t test. A p-value <0.05 396

was considered statistically significant. n.s. = not significant, * = significant (p ≤ 0.05), 397

** = highly significant (p ≤ 0.01), *** = very highly significant (p ≤ 0.001). Error bars 398

indicate the SD. 399

400

ACKNOWLEDGMENT 401

We thank Ron A. M. Fouchier (Erasmus Medical Center, Rotterdam, The Netherlands) 402

for providing the MERS-CoV EMC strain, and John Ziebuhr (University of Würzburg, 403

Würzburg, Germany) for providing the SARS-CoV Frankfurt 1 strain. We also thank 404

Aiko Fukuma and Hideki Tani (NIID, Japan) for valuable suggestions. This study was 405


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supported by a Grant-in-Aid for Scientific Research from the Japan Society for the 406

Promotion of Science (Grant No. 17K08868). 407

408

REFERENCES 409

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Biol. Chem. 284:15729–15738. 516

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FIGURE LEGENDS 541

Fig. 1. Comparison of three assays to quantify virus cell entry. 542

(A–C) Cell entry by pseudotyped or authentic MERS-CoV. Vero/TMPRSS2 cells in 543

96-well plates were infected with the serially diluted viruses indicated below. The 544

relationship between inoculated virus titer (x-axis) and data values (average of two 545

experiments) for each assay (y-axis) is shown. 546

(A) VSV-ΔG/GFP-MERS-S. GFP-positive cells were counted at 20 h post-infection. 547

(B) VSV-ΔG/Luc-MERS-S. Luciferase activity in cells was measured at 20 h 548

post-infection. 549

(C) Authentic MERS-CoV. The amount of viral mRNA in cells at 6 h post-infection was 550

measured by real-time PCR. 551

(D–F) Effect of a furin inhibitor on cell entry by pseudotyped or authentic MERS-CoV. 552

Vero/TMPRSS2 cells were inoculated with viruses in the presence or absence of the furin 553

inhibitor dec-RVKR-CMK. Virus entry was measured using appropriate assays. 554

(D) Effect of dec-RVKR-CMK on cell entry by VSV-ΔG/GFP-MERS-S. Cells were 555

inoculated with 103 infectious units of VSV-ΔG/GFP-MERS-S (MOI = 0.01). 556

GFP-positive cells were counted at 20 h post-infection (n = 4). 557

(E) Effect of dec-RVKR-CMK on cell entry by VSV-ΔG/Luc-MERS-S. Cells were 558

inoculated with 104 infectious units of VSV-ΔG/Luc-MERS-S (MOI = 0.1). Luciferase 559

activity in cells was measured at 20 h post-infection (n = 4). Data are presented on a 560

linear (left panel) and a logarithmic (right panel) scale. 561

(F) Effect of dec-RVKR-CMK on cell entry by authentic MERS-CoV. Cells were 562

inoculated with 105 infectious units of authentic MERS-CoV (MOI = 1). The amount of 563


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viral mRNA in cells at 6 h post-infection was measured by real-time PCR (n = 4). 564

A two-tailed Student t test was used to analyze statistical significance. n.s. = not 565

significant, * = significant (p ≤ 0.05), ** = highly significant (p ≤ 0.01), *** = very 566

highly significant (p ≤ 0.001). Error bars indicate the standard deviation (SD). ND = not 567

detected. 568

569

Fig. 2. Effect of a furin inhibitor on MERS-CoV entry into Calu-3 cells. 570

Calu-3 human bronchial epithelial cells were pretreated for 30 min with increasing 571

concentrations (0–100 μM) of the furin inhibitor dec-RVKR-CMK. E64d (10 μM), 572

camostat (10 μM), or a combination of both was used as a comparison control. The cells 573

were then infected with 105 plaque-forming units (pfu) of MERS-CoV (MOI = 1) in the 574

presence of inhibitor. The amount of viral mRNA in Calu-3 cells at 6 h post-infection was 575

measured by real-time PCR (n = 4). A two-tailed Student t test was used to analyze 576

statistical significance, as described in the legend to Figure 1. Error bars indicate the SD. 577

578

Fig. 3. Comparison of transcripts in LoVo cells. 579

(A) Expression of mRNA in Calu-3, LoVo, and Huh-7 cells. Total cellular RNA (0.1 μg) 580

was evaluated for expression of GAPDH, DPP4, furin, cathepsin L, TMPRSS2, and HAT 581

transcripts using real-time PCR (n = 4). ND, no transcripts were detected. 582

(B) Electropherograms of furin cDNA. The mRNAs isolated from Calu-3 and LoVo cells 583

were reverse-transcribed and amplified using a thermal cycler and used for DNA 584

sequencing. 585

(C) Viral yield in LoVo cells. Confluent Calu-3 cells and LoVo cells were grown in 586

96-well plates and infected with MERS-CoV at a MOI of 0.001 for 24 h. Cell-free 587

supernatants were harvested, and infectious viral titers were measured in a standard 588

plaque assay using Vero/TMPRSS2 cells (n = 4). 589


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A two-tailed Student t test was used to analyze statistical significance, as described in the 590

legend to Figure 1. Error bars indicate the SD. 591

592

Fig. 4. Effect of furin inhibitor on MERS-CoV and SARS-CoV entry into LoVo cells. 593

(A) Effect of inhibitors on MERS-CoV entry. LoVo cells were pretreated for 30 min with 594

the furin inhibitor dec-RVKR-CMK (concentration, 0–100 μM). E64d (10 μM), camostat 595

(10 μM), or a combination of both was used as a comparison control. The cells were then 596

infected with 105 pfu of MERS-CoV (MOI = 1) in the presence of inhibitor. The amount 597

of viral mRNA in LoVo cells at 6 h post-infection was measured by real-time PCR (n = 598

4). 599

(B) Effect of inhibitors on SARS-CoV entry. SARS-CoV was used instead of 600

MERS-CoV; all experiments were carried out as described in panel A. 601



604

Fig. 5. Effect of mutations in the furin cleavage site of the MERS-CoV S protein. 605

(A) Cleavage of the S protein on virions. Wild-type (wt) and mutant MERS-CoV lacking 606

furin cleavage sites within the S protein (generated in Vero/TMPRSS2 cells) were 607

subjected to western blot analysis with an anti-S polyclonal antibody. 608

(B) Cleavage of S protein in cells. The wt and mutant viruses shown in panel A were used 609

to infect Vero/TMPRSS2, Huh-7, and LoVo cells, and cell lysates prepared at 20 h 610

post-infection were subjected to western blot analysis. 611

(C) Entry of mutant viruses into LoVo and Huh-7 cells. The cells were infected with 104 612

pfu of MERS-CoV (MOI = 0.1). The amount of viral mRNA in cells at 6 h post-infection 613

was measured by real-time PCR (n = 4). 614

(D) Effect of mutations on virus entry. LoVo cells were pretreated for 30 min with the 615


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furin inhibitor dec-RVKR-CMK (concentration at 100 μM). Then, cells were infected 616

with 104 pfu of MERS-CoV (MOI = 0.1) in the presence of inhibitor. The amount of viral 617

mRNA in cells at 6 h post-infection was measured by real-time PCR (n = 4). Data are 618

expressed as the -fold change in viral mRNA levels relative to that in the absence of 619

dec-RVKR-CMK. 620



623

Fig. 6. S protein activation by exogenous furin. 624

(A) Activity of commercial furin. The enzymatic activity of recombinant human furin 625

used in the experiment below was confirmed using the fluorescent substrate 626

BOC-RVRR-AMC. 627

(B) Exogenous furin does not trigger cell entry by virus. LoVo cells in 96-well plates 628

were inoculated with MERS-CoV (MOI of 0.1) for 30 min and then treated with furin 629

(2000 units/ml) or trypsin (10 μg/ml) at 37°C for 10 min. Cells were then cultured at 630

37°C for 6 h. The amount of viral mRNA was measured by real-time PCR (n = 4). A 631

two-tailed Student t test was used to analyze statistical significance, as described in the 632


(C) Exogenous furin does not induce syncytium formation. LoVo cells infected with 634

MERS-CoV (MOI of 0.01) were cultured for 20 h and then treated with furin (2000 635

units/ml) or trypsin (10 μg/ml) for 5 h. 636

(D) Cleavage of S protein by exogenous furin. MERS-CoV propagated in Vero cells was 637

treated with the indicated concentrations of trypsin or furin at 37°C for 60 min. Western 638

blot analysis was performed using an anti-S polyclonal antibody. 639

640

Fig. 7. Timing of inhibitor addition to block entry of MERS-CoV into cells. 641


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(A) Dec-RVKR-CMK (100 μM), camostat (10 μM) plus E64d (10 μM), or lopinavir (30 642

μM) was added to LoVo cells (for 30 min on ice) at the indicated times before and after 643

MERS-CoV inoculation. The amount of viral mRNA in cells at 6 h post-infection was 644

measured by real-time PCR (n = 1). 645

(B) Calu-3 cells were used instead of LoVo cells; all experiments were carried out as 646

described in panel A (excluding lopinavir treatment) (n = 1). 647

648

Fig. 8. Effect of furin inhibitor on commercial proteases and cell surface TMPRSS2. 649

(A) Inhibitory effect of dec-RVKR-CMK on commercial proteases. Degradation products 650

of fluorescein-labeled casein generated by treatment with commercial proteases, 651

cathepsin L (20 μg/ml), cathepsin B (20 μg/ml), trypsin (50 μg/ml), papain (0.3 units/ml), 652

proteinase K (0.3 units/ml), dispase (3 units/ml), elastase (200 μg/ml), and chymotrypsin 653

(10 μg/ml), at 37°C for 30 min in the presence of dec-RVKR-CMK (serially diluted 654

10-fold) were quantified by fluorometry (n = 3). Data are expressed as an average 655

percentage relative to that in the absence of dec-RVKR-CMK. N, no inhibitor treatment. 656

(B) E64d and bafilomycin A1 do not inhibit commercial proteases. E64d or bafilomycin 657

A1 was used instead of dec-RVKR-CMK; all experiments were carried out as described 658

in panel A (proteases tested were trypsin, chymotrypsin, and elastase). 659

(C) Inhibitory effect of dec-RVKR-CMK against TMPRSS2, as assessed in the FFWO 660

assay. A high titer of MERS-CoV (MOI = 10) was adsorbed onto Vero/TMPRSS2 or Vero 661

cells (on ice for 1 h). Cells were then incubated at 37°C in the presence of the inhibitor. 662

After 5 h, cells were fixed and stained with crystal violet. 663


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Figure 1

VSV-ΔG/Luc-MERS-S VSVΔG/GFP-MERS-S

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C T T T G G A C

C T T T T G G A

Calu-3

LoVo

C G A G T G G G

C G A G C G G G

1286 1639

C T T T T G G A

C T T T T G G A

C G A G T G G G

C G A G T G G G

1635-1642 1282-1289

furin

Figure 3

0

1

2

3

4

5

6

0 h

24

h

0 h

24

h

LoVo Calu-3

log

10P

FU

/10

0 µ

l

B A C


IVE

RS

ITY

OF

NE

W E

NG

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A

Co

pie

s o

f M

ER

S-C

oV

su

bg

en

om

ic R

NA

(lo

g10)

MERS-CoV/LoVo cells

**

n.s.

**

ND

***

***

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0.8

4

20

10

0

E6

4d

Ca

mo

sta

t 0

dec-RVKR-CMK

(µM)

Ca

mo

sta

t +

E4

6d

ND

Co

pie

s o

f S

AR

S-C

oV

su

bg

en

om

ic R

NA

(lo

g10)

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0.8

4

20

10

0

E6

4d

Ca

mo

sta

t 0

dec-RVKR-CMK

(µM)

Ca

mo

sta

t +

E4

6d

**

n.s. *

***

**

*

n.s.

SARS-CoV/LoVo cells

B

Figure 4


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LAN

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1/1000

1/100

1/10

1

10

No-treatment

100 µM dec-RVKR-CMK

Su

bg

en

om

ic R

NA

[-fo

ld c

ha

ng

e]

C

mutations at the predicted

furin cleavage site

wt

*** *** *** ***

Figure 5

250

150

100

75

50

kD

wt E

MC

R

748S

R

884S

R

748S

/R884S

N

o v

iru

s

Vero/TMPRSS2

wt E

MC

R

748S

R

884S

R

748S

/R884S

N

o v

iru

s

wt E

MC

R

748S

R

884S

R

748S

/R884S

N

o v

iru

s

S

Huh-7 LoVo

250

150

100

75

50

kD

S

wt E

MC

R

748S

R

884S

R

748S

/R884S

N

o v

iru

s

GAPDH

A B cell lysate culture medium

Vero/TMPRSS2

mutations at the predicted

furin cleavage site

wt

LoVo cells

1 2 3 4 5

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

D

1

2

3

4

5

Huh-7 LoVo

n.s.

n.s.

n.s.

Co

pie

s o

f M

ER

S-C

oV

su

bg

en

om

ic R

NA

(lo

g10)


IVE

RS

ITY

OF

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W E

NG

LAN

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Figure 6

2000 units/ml furin

0

200

400

600

800

1000

1200

units/ml

Flu

ore

scence in

tensity

0

0.0

1

0.1

1

250

150

100

75

50

37 25

kD

D

A LoVo cells

Co

pie

s o

f su

bgenom

ic

vira

l R

NA

(lo

g1

0)

2.5

3.0

3.5

4.0

4.5

**

n.s.

LoVo cells

No

tr

eatm

ent

1 µ

g/m

l tr

ypsin

2000 u

nits/m

l

furin

C

B

-

S

by trypsin

by trypsin

by furin

by furin

trypsin (µg/ml) 0

0.0

1

0.1

1

1 2 3 4 5 6 7 8


IVE

RS

ITY

OF

NE

W E

NG

LAN

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Calu-3 cells

Co

pie

s o

f M

ER

S-C

oV

su

bg

en

om

ic R

NA

(lo

g10)

Figure 7

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 LoVo cells

-60 -30 0 30 60

Time of inhibitor addition

(minutes)

-60 -30 0 30 60

Time of inhibitor addition

(minutes)

120 240 360

dec-RVKR-CMK

E64d+Camostat

Lopinavir

A B

Co

pie

s o

f M

ER

S-C

oV

su

bg

en

om

ic R

NA

(lo

g10)

360


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RS

ITY

OF

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W E

NG

LAN

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E64d

0

20

40

60

80

100

120

N -1 0 1 2

Trypsin Chymo

Elastase

elastase

N -1 0 1 2

cathepsin L

0

20

40

60

80

100

120

N -1 0 1 2

cathepsin B

0

20

40

60

80

100

120

N -1 0 1 2

papain

N -1 0 1 2

trypsin

N -1 0 1 2

proteinase K

N -1 0 1 2

dispase

N -1 0 1 2

chymotrypsin

N -1 0 1 2

bafilomycin A1

0

20

40

60

80

100

120

N -1 0 1 2

Inhibitor (log10 nM) dec-RVKR-CMK (log10 µM)

Pe

rce

nt

pro

tea

se

activity

A B

Pe

rce

nt

pro

tea

se

activity

DMSO dec-RVKR-CMK

- 100 µM 20 µM 10 µM

camostat

Vero/TMPRSS2

C

Figure 8

Inhibitor (log10 µM) dec-RVKR-CMK (log10 µM)

Vero

MERS-CoV No virus MERS-CoV No virus

DMSO

-

DMSO

-

DMSO

-


IVE

RS

ITY

OF

NE

W E

NG

LAN

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Documents

2018 Middle East respiratory syndrome coronavirus spike protein is not activated directly by cellular furin during viral