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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 Kamitanic 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 matuyama nih go jp 13 14 JVI Accepted Manuscript Posted Online 18 July 2018 J Virol doi 10 1128 JVI 00683 18 Copyright 2018 American Society for Microbiology All Rights Reserved on July 20 2018 by UNIVERSITY OF NEW ENGLAND http jvi asm org Downloaded from 2 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 on July 20 2018 by UNIVERSITY OF NEW ENGLAND http jvi asm org Downloaded from 3 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 on July 20 2018 by UNIVERSITY OF NEW ENGLAND http jvi asm org Downloaded from 4 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 M 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 G GFP MERS S 2 the 88 same virus in which the GFP gene was replaced by the luciferase Luc gene 89 VSV G 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 G GFP MERS S was narrow 1 3 log10 GFP cell 93 on July 20 2018 by UNIVERSITY OF NEW ENGLAND http jvi asm org Downloaded from 5 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 M on viral entry into cells 98 Infection with VSV G 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 G 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 G 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 M suppressed virus entry by 117 100 fold similar results were observed after treatment with 20 M dec RVKR CMK 118 E64d had little effect on virus entry suggesting that MERS CoV mainly uses the 119 on July 20 2018 by UNIVERSITY OF NEW ENGLAND http jvi asm org Downloaded from 6 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 M 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 on July 20 2018 by UNIVERSITY OF NEW ENGLAND http jvi asm org Downloaded from 7 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 on July 20 2018 by UNIVERSITY OF NEW ENGLAND http jvi asm org Downloaded from 8 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 g 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 M dec RVKR CMK or a mixture of 10 M E64d and 10 M 196 camostat E64d camostat during infection with MERS CoV Lopinavir which targets 197 on July 20 2018 by UNIVERSITY OF NEW ENGLAND http jvi asm org Downloaded from 9 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 M 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 on July 20 2018 by UNIVERSITY OF NEW ENGLAND http jvi asm org Downloaded from 10 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 M suppressed cell cell fusion completely as did 232 camostat 10 M 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 on July 20 2018 by UNIVERSITY OF NEW ENGLAND http jvi asm org Downloaded from 11 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 proteas