VIROLOGY 214 453 463 1995 Mutational Analysis of the Murine Coronavirus Spike Protein Effect on Cell to Cell Fusion EVELYNE C W BOS LEO HEIJNEN WILLEM LUYTJES and WILLY J M SPAAN 1 Department of Virology Institute of Medical Microbiology Faculty of Medicine Leiden University 2300 AH Leiden The Netherlands Received July 5 1995 accepted October 4 1995 The spike S protein of murine coronavirus strain A59 MHV A59 is a type I membrane protein that induces membrane fusion In this study we have analyzed the role of two domains in the S protein on fusion The 180 kDa mature S protein is partially cleaved into two 90 kDa subunits during transport to the plasma membrane We have identified several amino acids that are important for cleavage of S and we show that cleavage is not strictly required for fusion However the level of cleavage seems to influence the fusion kinetics After introduction of an arginine at position P 2 to mimick the MHV JHM cleavage site full cleavage of the spike protein was obtained Further we analyzed the effect of mutations in the transmem brane TM domain of the S protein Maturation and cell surface expression of the mutant proteins were not affected and all proteins became acylated The mutant in which the predicted transmembrane domain was shortened did not induce syncytia From a group of mutants in which several conserved cysteines in the TM domain had been replaced by serines one was unable to induce syncytia another showed delayed syncytia formation and the third mutant induced syncytia as did the wild type protein The potential role of the transmembrane domain in fusion is discussed q 1995 Academic Press Inc INTRODUCTION and modified giving rise to an almost endo H resistant 180 to 200 kDa protein A distinctive feature among viral Membrane fusion is a key event in the replication cycle fusion proteins is that irrespective of the pH optimum of enveloped viruses During penetration of the host cell some must undergo cleavage activation e g hemagglu the viral membrane fuses with either the plasma mem tinin of influenzavirus gp160 of HIV whereas others are brane or endosomal membrane resulting in the release fusogenic in an uncleaved form e g G protein of rhab of the viral genome into the cytosol of the infected cell doviruses reviewed by White 1990 Whether fusion occurs at the endosomal membranes or In several coronaviruses such as infectious bronchitis at the plasma membrane depends on the pH at which virus IBV murine coronavirus MHV and bovine coro the viral fusion protein is in its fusogenic conformation navirus BCV the S proteins are cleaved reviewed by Fusion at the plasma membrane occurs when this con Spaan et al 1988 Proteolytic cleavage of S resulting formation is reached at neutral pH Fusion can also be in S1 and S2 by host cell proteases occurs as a late induced when the fusion protein is expressed at the cell step in transport The 180 kDa S protein of MHV strain surface of infected cells leading to cell to cell fusion A59 is only partially cleaved whereas cleavage of strain In this case large syncytia are formed which can add JHM is almost complete In 17Cl1 cells however almost significantly to the intracellular spread of these viruses no cleavage of MHV A59 S protein was observed Frana reviewed by White 1990 et al 1985 Trypsin treatment of MHV A59 virus purified Membrane fusion in coronavirus infected cells is me from 17Cl1 cells resulted in cleavage of S between amino diated by the spike S protein and takes place at neutral acid residues 717 and 718 The sequence at the cleavage pH Sturman and Holmes 1985 Cell surface expression site is RRAHR f SVS Luytjes et al 1987 of S in infected or recombinant S expressing cells results In the presence of leupeptin an inhibitor of serine and in the formation of extensive syncytia Vennema et al thiol proteases the formation and size of syncytia was 1990 The S protein is a glycosylated Niemann and delayed and reduced respectively Frana et al 1985 Klenk 1981 and palmitine acylated van Berlo et al Cell fusion activity of MHV A59 virions purified from 1987 Niemann and Klenk 1981 Ricard and Sturman 17Cl1 cells required the addition of trypsin Sturman et 1985 Schmidt 1982 type I membrane protein During al 1985 These data strongly suggested that only A59 transport of the trimerized Cavanagh et al 1983 Delmas virions with cleaved S are fusogenic and are supported and Laude 1990 precursor protein through the Golgi by the observations of Gombold et al 1993 who have apparatus the high mannose side chains are trimmed isolated cleavage negative MHV A59 variants which are fusion negative However it has been shown recently that recombinant S proteins of MHV JHM in which the 1 To whom correspondence and reprint requests should be ad dressed Fax 31 71263645 E mail azruviro rulcri LeidenUniv nl cleavage site was mutated were still able to induce cell 453 0042 6822 95 12 00 Copyright q 1995 by Academic Press Inc All rights of reproduction in any form reserved m4655 7604 11 15 95 08 12 22 viral AP Virology454 BOS ET AL to cell fusion Stauber et al 1993 Taguchi 1993 The and m7 were constructed using Kunkel mutagenesis on the BamHI SphI fragment of pTugMS cloned in the heptad repeat region just upstream of the transmem brane anchor De Groot et al 1987 has also been con pBluescript KS 0 vector Mutants m40 m41 m42 and m43 were constructed by Kunkel mutagenesis on the KpnI nected with fusion Gallagher et al 1991 Grosse and Siddell 1994 SalI fragment of pTugMS in pBluescript KS 0 The mu tated fragments were sequenced and subsequently used The membrane anchoring sequence is another part of the fusion protein that has been associated with fusion to replace the corresponding nonmutated fragment in pTugMS activity in several viruses Ragheb and Anderson 1994 Rasile et al 1993 Mulligan et al 1992 The coronavirus Virus infection S protein is anchored in the lipid bilayer by a large 34 45 aa transmembrane TM domain which is defined by Confluent monolayers of L cells were infected with the bordering charged amino acid residues reviewed by MHV A59 or MHV JHM in PBS DEAE supplemented Boyd and Beckwith 1990 At the start of the TM domain with 3 FCS at a multiplicity of infection m o i of 10 in all coronavirus S protein sequences studied to date a After absorption for 1 hr at 377 virus was removed and highly conserved stretch of seven large hydrophobic cells were cultured in DMEM supplemented with 3 FCS amino acids is found The first part of the TM anchor is a hydrophobic region of about 20 22 amino acids fol Protein expression lowed by a cysteine rich hydrophillic region of 11 23 Mouse L cells 11 10 6 were seeded in 35 mm dishes amino acids Sixteen hours later the cells were infected with the T7 We have studied both the cleavage domain and the RNA polymerase expressing vaccinia recombinant TM anchor of coronavirus MHV A59 Here we demon vTF7 3 at a m o i of 5 At 1 5 hr postinfection the cells strate by site directed mutagenesis that the endogenous were transfected with 1mg DNA per 35 mm plate using protease yet to be identified cleaves the MHV A59 S the lipofectine procedure Gibco BRL as described by protein at the RAHR f motif Several amino acid residues the manufacturer important for cleavage were identified We also demon Before labeling the cells were incubated for 30 min strate that cleavage of S is not a prerequisite for cell in DMEM deficient in methionine Subsequently the cells fusion Furthermore we show that the transmembrane were metabolically labeled with 100 mCi 35 S labeled anchor of the MHV A59 S protein is required for fusion amino acids Expre 35 SS label NEN in medium lacking methionine or with 100 mCi 3 H palmitic acid NEN in MATERIALS AND METHODS complete medium When a pulse chase experiment was performed the radioactive medium was replaced with Cells and viruses DMEM containing four times the normal concentration Mouse L cells 17Cl1 and rabbit RK13 cells were of methionine Cells were lysed in RIPA buffer 150 mM grown in Dulbecco s modified Eagle s medium DMEM NaCl 1 0 NP 40 0 5 DOC 0 1 SDS 50 mM Tris pH Gibco containing 10 fetal bovine serum Vaccinia virus 8 0 and 2 mM PMSF The lysate was centrifuged at 47 vTF7 3 stocks kindly provided by Dr B Moss were for 10 min at 13 000 rpm to remove the nuclei and cell grown on RK13 cells MHV A59 stocks were grown on debris Immunoprecipitations were performed on the su 17Cl1 cells MHV JHM was kindly provided by Dr S G pernatant using a mixture of S specific monoclonal anti Siddell bodies J7 6 and J1 4 Fleming et al 1983 and wa3 1 Gilmore et al 1987 Antibody A1 kindly provided by Dr Site directed mutagenesis H Wege was used to immunoprecipitate the MHV JHM S protein After an overnight incubation at 47 5 0m l Pan The complete MHV A59 spike gene MHV S has been sorbin cells Calbiochem La Jolla CA and KCl to a cloned sequenced and reconstructed previously final concentration of 0 5 M were added followed by an Luytjes et al 1987 Vennema et al 1990 The full length incubation for 1 hr at 47 After washing the samples three S gene was cloned as a BamHI fragment between a times in RIPA they were boiled in Laemmli sample buffer T7 promoter and terminator in the BamHI site of the for 2 min Laemmli 1970 Protein samples were endo expression vector pTUG31 Vennema et al 1991 From H treated as described by Vennema et al 1990 The this clone pTugMS a 662 bp KpnI SphI fragment was immune precipitates were analyzed by SDS PAGE on isolated and ligated in KpnI and SphI digested M13 10 gels mp18 DNA The resulting M13 template was used for site directed mutagenesis described by Kunkel Kunkel Indirect immunofluorescence et al 1987 Mutant m1 was designed after mutant 4 of paramyxovirus SV 5 Paterson et al 1989 Amino acid Cells were cultured on glass coverslips in 35 mm wells and infected and transfected as described above Cov substitutions in mutants m3 m4 m5 and m6 were de signed to mimic the influenza HA cleavage mutants de erslips were removed at set intervals and fixed in 4 paraformaldehyde to monitor cell surface expression of scribed by Kawaoka and Webster 1988 Mutants m2 m4655 7604 11 15 95 08 12 22 viral AP Virology455 MHV A59 FUSION REQUIREMENTS lanes 6 to 7 The lack of cleavage resulted in a higher level of 180 kDa protein compared to wt S transfected cells Surface immunofluoresence was detected with wt m1 and m2 S protein data not shown These results strongly suggest that an endogenous protease cleaves the MHV A59 S protein at the same position as trypsin does The absence of cleavage of mutant m2 also implies an important role for the arginine residue at position P 1 We also tested whether the transition of the 150 kDa precursor protein to the gp180 90 mature form could be improved by incubating the vaccinia virus infected and DNA transfected cells at 327 as has been suggested by Marquardt and Helenius 1992 No significant difference FIG 1 Mutations introduced upstream of the trypsin cleavage site of MHV A59 S protein For the mutants only mutated residues are in the maturation of gp150 at the two temperatures was depicted Asterisks represent deleted amino acids Numbers above the observed and therefore all subsequent experiments were amino acids indicate the position of that particular amino acid relative carried out at 377 to the trypsin cleavage site The cleavage phenotypes of the mutants Next we examined in more detail the sequence re is shown in the table at the right quirements for cleavage of the S protein Four additional mutants were generated three containing single amino S proteins Cells were washed several times in PBS sup acid changes at position P 5 P 4 o rP 2 of the cleavage site plemented with 10 mM glycine reacted with a 1 5 diluted and one having a deletion of the arginine residue at monoclonal antibody mix of J7 6 J1 4 and wa3 1 for 30 position P 5 Fig 1 The effect of these mutations on the min and developed with 1 50 diluted goat anti mouse proteolytic cleavage of S was tested Immunoprecipitated FITC conjugated mAb Dako proteins were endo H treated as described under Mate rials and Methods In all cases the most abundant S RESULTS protein was the endo H sensitive 150 kDa core protein Mutant m5 in which the histidine residue is replaced by Mutagenesis of the cleavage site of the MHV A59 a glutamine was cleaved at a level comparable to the spike protein wt S protein Fig 3 lanes 7 and 8 Substitution of the arginine residue at position P 5 by glycine m3 had only Protein sequencing of the trypsin generated N termi nus of the MHV A59 S2 subunit has revealed the position a limited effect on cleavability Fig 3 lanes 3 and 4 When the arginine residue at position P 4 was replaced of the cleavage site Luytjes et al 1987 To determine whether the trypsin cleavage site is also recognized by by a threonine residue no cleaved S could be detected Neither did we observe any cleavage in which the argi the endogenous protease that cleaves S during transport to the cell surface we have constructed several mutant nine residue at position P 5 was deleted Fig 3 lanes 5 6 9 10 The presence of the endo H resistant 180 kDa m S genes In m1 the sequence encoding the peptide R 5 R 4 A 3 H 2 R 1 which is located at the C terminus of S protein in both m4 and m6 transfected cells clearly demonstrates that the cleavage negative phenotype of trypsin cleaved S1 Fig 1 has been deleted whereas in the second mutant m2 the arginine residue at the P 1 position has been replaced by a glycine residue To study the processing of both mutants L cells were infected with the vaccinia T7 RNA polymerase recombinant vTF7 3 and transfected with the wild type wt m1 and m2 S expressing plasmids After metabolic labeling for 1 hr followed by a chase of 2 hr the cells were lysed and S protein was immunoprecipitated using a mixture of three different S specific monoclonal antibodies and analyzed by SDS PAGE Fig 2 In both MHV infected cells see below and in cells transfected with the wt S encoding plasmid S was mostly present as a 150 kDa core protein In addition the FIG 2 Cleavage pattern of wt and mutant S proteins L cells tran siently expressing wt or mutant spike proteins were pulse labeled with mature 180 and 90 kDa species could be detected Fig 100 mCi Expres 35 SS label from 5 to 6 hr posttransfection lanes 2 4 2 lane 4 During the chase period the intensity of the 6 8 and subsequently chased for 2 hr lanes 3 5 7 9 Spike proteins gp 180 band increased In m1 and m2 transfected cells were immunoprecipitated using a mix of anti S monoclonal antibodies only the 150 kDa precursor and the mature 180 kDa S and analyzed as described under Materials and Methods Marker lane proteins could be detected Even after the 2 hr chase 1 mock transfected cells lanes 2 3 wt S lanes 4 5 m1 lanes 6 7 and m2 lanes 8 9 period cleavage products could not be observed Fig 2 m4655 7604 11 15 95 08 12 22 viral AP Virology456 BOS ET AL converted into the 90 kDa cleavage product during the chase period but no 180 kDa protein was detected Fig 4A lanes 7 and 8 Even after prolonged exposure the 180 kDa was not observed in JHM infected cells data not shown The different cleavage patterns of MHV A59 and MHV JHM S proteins are thus not host cell depen dent To test whether the difference in cleavability between the S proteins of JHM and A59 was dependent on the FIG 3 Acquisition of endo H resistance of wt and mutant S proteins amino acid at position P 2 of the cleavage site an A59 L cells expressing S were labeled with 100mCi Expres 35 SS label from 7 to 11 hr posttransfection Immunoprecipitated S proteins were incu mutant S protein was constructed in which the histidine bated at 377 for 16 hr without lanes 1 3 5 7 9 11 or with lanes 2 at position P 2 was replaced by an arginine residue The 4 6 8 10 12 endo H and analyzed as described Cells were sequence of the cleavage domain of this mutant m7 transfected with wt S lanes 1 and 2 m3 lanes 3 and 4 m4 lanes 5 Fig 1 is thus identical to the JHM cleavage site After a and 6 m5 lanes 7 and 8 m6 lanes 9 and 10 or m1 lanes 11 and 2 hr chase the endo H resistant 180 kDa product was 12 S pS and S1 S2 at the left indicate the positions of the 180 kDa the 150 kDa precursor protein and the cleaved S1 and S2 subunits observed in the lysate obtained from wt S transfected respectively before endo H treatment After endo H treatment the pro cells only The 180 kDa band could not be detected in teins shift to the positions indicated at the right S H p S H and S1 S2 H the m7 lysates Fig 4B lane 8 The 90 kDa forms of both the wt and m7 S protein were clearly present These results indicate that the S protein of A59 acquires high both mutant S proteins is not due to the lack of transport cleavability when an arginine residue is introduced at of the endo H sensitive gp150 protein position P 2 instead of a histidine residue The amino acid at position P 2 is therefore important in determining the Introduction of a dibasic cleavage site results in efficiency of cleavage of MHV S complete processing of the A59 S protein As described above the mature 180 kDa wt S protein Induction of cell to cell fusion by cleavage site of MHV A59 is only partially cleaved In strain MHV JHM mutants the motif RRARR is present at the cleavage site and in JHM only the cleaved form of the mature S protein is The ability of mutant S proteins to induce cell to cell fusion was examined in vTF7 infected mouse L cells observed in the virion and in infected cells It has been suggested Daya et al 1989 Mizzen et al 1983 that Cells expressing the wt and the mutant S proteins were fixed at 4 and 8 hr posttransfection and S protein expres the differences in cleavage efficiencies are host related Because cleavage of A59 and JHM S proteins has never sion was detected by indirect immunofluorescence The wt S protein induced syncytia that were readily visible been compared in the same cell line we have analyzed the maturation of the MHV A59 and MHV JHM S proteins after 4 hr and increased in time Cleavage negative S mutants were expressed at the surface but were delayed in L cells In the A59 infected cell lysate most of S was present as the endo H sensitive 150 kD form Fig 4A in syncytium induction Results for wt S and m2 are shown in Fig 5 The results for the other cleavage nega Upon a 2 hr chase part of the 150 kDa S protein was converted into the 180 kDa form and some protein was tive mutants were similar to m2 whereas m3 and m5 were almost indistinguishable from wt data not shown chased to 90 kDa Fig 4A lanes 5 and 6 For MHV JHM a considerable amount of 150 kDa protein S protein was Thus cleavage of the S protein is not a prerequisite FIG 4 Effect on cleavage efficiency of introduction of the JHM cleavage site into the MHV A59 spike protein A Comparison of cleavage of the MHV A59 and MHV JHM S proteins Immunoprecipitation of S from L cells that were infected with either MHV A59 lanes 1 2 5 6 or MHV JHM lanes 3 4 7 8 at a m o i of 10 Cells were labeled with 100mCi Expres 35 SS label from 6 to 7 hr posttransfection lanes 1 2 3 4 and chased for 2 hr lanes 5 6 7 8 Proteins were immunoprecipitated using a mAb anti S mix A59 or anti S monoclonal antibody A1 JHM Part of the lysate was subsequently treated with endo H lanes 2 4 6 8 B Comparison of the cleavage efficiency between wt S and m7 L cells expressing wt S lanes 1 2 5 6 or m7 lanes 3 4 7 8 were pulse labeled for 1 hr t 5 to 6 lanes 1 to 4 and chased for 2 hr lanes 5 to 8 Immunoprecipitations were performed using an anti S mAb mix Proteins were subsequently incubated at 377 for 16 hr without lanes 1 3 5 7 or with lanes 2 4 6 8 endo H Positions of the different S forms are indicated as described in the legends of Fig 3 m4655 7604 11 15 95 08 12 22 viral AP Virology457 MHV A59 FUSION REQUIREMENTS FIG 5 Cell surface expression of S proteins on L cells using the vaccinia expression system After vTF7 3 infection and DNA transfection cells were fixed using paraformaldehyde at 4 and 8 hr posttran