2014 Identification and Characterization of a Proteolytically Primed Form of the Murine Coronavirus Spike Proteins after

2014·1.77 MB·English

Identification and Characterization of a Proteolytically Primed Form
of the Murine Coronavirus Spike Proteins after Fusion with the
Target Cell
OliverWicht,ChristineBurkard,CornelisA.M.deHaan,FrankJ.M.vanKuppeveld,PeterJ.M.Rottier,BerendJanBosch
VirologyDivision,DepartmentofInfectiousDiseasesandImmunology,FacultyofVeterinaryMedicine,UtrechtUniversity,Utrecht,TheNetherlands
D
ABSTRACT o
w
Envelopedvirusescarryhighlyspecializedglycoproteinsthatcatalyzemembranefusionunderstrictspatialandtemporalcon- n
trol.Topreventprematureactivationafterbiosynthesis,viralclassIfusionproteinsadoptalockedconformationandrequire lo
a
proteolyticcleavagetorenderthemfusion-ready.Thisprimingstepmayoccurduringvirusexitfromtheinfectedcell,inthe d
e
extracellularmilieuorduringentryatorinthenexttargetcell.Proteolyticprocessingofcoronavirusspike(S)fusionproteins d

duringvirusentryhasbeensuggestedbutnotyetformallydemonstrated,whilethenatureandfunctionalityoftheresultingsub- fr
o
unitisstillunclear.Weusedaprototypecoronavirus—mousehepatitisvirus(MHV)—todevelopaconditionalbiotinylation m
assaythatenablesthespecificidentificationandbiochemicalcharacterizationofviralSproteinsonvirionsthatmediatedmem- h
t
branefusionwiththetargetcell.WedemonstratethatMHVSproteinsareindeedcleaveduponvirusendocytosis,andweiden- tp
:
tifyanovelprocessingproductS2*withcharacteristicsofafusion-activesubunit.Theprecisecleavagesiteandtheenzymesin- //
jv
volvedremaintobeelucidated. i.
a
s
m
IMPORTANCE
.
Virusentrydeterminesthetropismandisacrucialstepintheviruslifecycle.Wedevelopedanapproachtocharacterizestruc- o
r
g
turalcomponentsofvirusparticlesafterenteringnewtargetcells.Aprototypecoronaviruswasusedtoillustratehowthevirus /

o
fusionmachinerycanbecontrolled. n

A
Enveloped viruses must fuse their envelope with a target cell Coronavirus(CoV)entryismediatedbythespike(S)protein, ug
u
membranetogetaccesstohostcellsanddelivertheirgenetic an exceptionally large glycoprotein of approximately 1,200 to s
t
information. They carry specialized surface glycoproteins that 1,450aminoacidresiduesinlengththatcomprisesthecanonical 1
mediateattachmenttoandfusionwiththehostmembrane.Viral structuralfeaturesofclassIfusionproteinsandsharesthetypical 1
,

fusionproteinscangenerallybedividedintothreedistinctclasses fusionmechanism(11).ThetrimericSproteinscharacteristically 2
0
accordingtotheirmolecularorganizationandfusionmechanism decoratetheextracellularvirusparticlesandtwosubunitsofsim- 1
5
(1).ClassIfusionproteinssuchastheinfluenzavirushemagglu- ilarsizecanbedistinguished.TheN-terminalS1subunitcontains
b
tininandthehumanimmunodeficiencyvirusEnvoccurasho- the receptor-binding domain, while the C-terminal S2 subunit y

motrimericglycoproteinsthatareorientedperpendiculartothe comprisesthefusionmachinery,includingaputativeFP,HRre- U
n
viralmembraneandcontaintypicalstructuralelements,including gions,andtransmembranedomain. iv
areceptor-bindingdomain,heptadrepeat(HR)regions,anam- SomeCoVSproteinsarecleavedattheS1/S2junctionduring o
f
phipathicfusionpeptide(FP),andaC-terminaltransmembrane biogenesisbyfurin(-like)proteases,butmanyCoVslackafurin M
domain(2).Thesefusionproteinsalsofeatureacommonfusion cleavage site at the S1/S2 junction and hence carry uncleaved S is
s
mechanism(3).Initialconformationalrearrangementstriggered proteinintheirvirions(12).Othercellularproteaseshavebeen o
bycuessuchasreceptorbindingorlowpHleadtotheexposure reported to cleave CoV S proteins, but these proteases are only ur
and insertion of the FP into the target membrane. Subsequent availableuponattachmentorduringtheuptakeofvirionsbythe i A
structural reorganization pulls the two membranes together to nexttargetcells(13).TheinfectionofsomeCoVscanbeblocked t
S
achievefusion.ThefreeenergyisprovidedbytheSproteinsand by protease inhibitors, thereby underlining the importance of t
L
releasedbyzippingupoftheheptadrepeatregionsintoanener- proteolytic activation that should render class I fusion proteins o
geticallyfavorable,stablesix-helixbundle(1).Topreventprema- intotheirfusion-competentform(6,14–16).Remarkably,acleav- uis
tureactivation,classIfusionproteinsareproducedinalocked ageattheS1/S2junctiondoesnotliberateaputativeFPattheN
conformation that needs proteolytic cleavage to acquire fusion terminusofS2(17).RatherthanattheS1/S2junction,cleavage
competence.CleavagetypicallyoccursjustupstreamoftheFPand
causesN-terminalliberationthereof(4).Furinorfurin-likepro-
teasesoftenprimethefusionproteinsintheproducercellbefore Received22November2013 Accepted20February2014
virions are released. Alternatively, the cleavage event can take Publishedaheadofprint19February2014
placeafterthereleaseofvirionsfromtheinfectedcell,i.e.,inthe Editor:S.Perlman
extracellularspaceoruponentryintonewhostcells(5–7).Pre- AddresscorrespondencetoBerendJanBosch,[email protected].
ventionoffusionproteincleavagebymutagenesisofthecleavage Copyright©2014,AmericanSocietyforMicrobiology.AllRightsReserved.
site, as well as by inhibition of cellular proteases, often renders doi:10.1128/JVI.03451-13
virusesnoninfectious(8–10).
May2014 Volume88 Number9 JournalofVirology p.4943–4952 jvi.asm.org 4943
Wichtetal.
can occur at alternative positions within the S2 domain of the ines.MHVS2*-BAPSproteincarriestwopointmutations—R867Sand
proteintopromotethefusioncompetence.Suchalternativecleav- R869S—thatsubstitutetheargininesattheputativeS2=cleavagesiteby
agesiteshavebeendescribedwithintheS2subunitfortheSpro- serines.
teinsofsevereacuterespiratorysyndromecoronavirus(SARS)- Generationofstablecelllines.ThepQCXIN-CCMplasmidencoding
CoV, mouse hepatitis virus (MHV), and infectious bronchitis theMHVreceptor,murinecarcinoembryonicantigen-relatedcelladhe-
virus(IBV)(16,18,19).Ingeneral,avarietyofputative,alterna- sionmolecule1a(CCM),wasgeneratedbycloningtheCCMgeneintothe
pQCXIN Moloney murine leukemia virus (MLV) packaging vector
tivecleavagesitesandcleavagetimingshavebeenreportedorsug-
(Clontech)(29).Likewise,thehumancodon-optimizedgeneencoding
gested for CoV, and yet the role of S protein cleavage remains
biotinproteinligase(BirA)withanN-terminalhemagglutininandFLAG
largelyundefined.
tag(thepUM376-BirAPCRtemplatewaskindlyprovidedbyV.Ogryzko)
Despiteextensiveresearchontheproteolyticrequirementsfor
wasclonedintothepQCXIPvector(Clontech),generatingthepQCXIP-
entry,theexactcleavagepositionwithintheCoVSproteingener-
BirApackagingvector(30).HEK-293T,HeLa,andVero-CCL81celllines D
atingthefusogenicsubunithasbeendifficulttopredict,andthe o
expressingtheCCMreceptorweremadeaftertransductionwithvesicular
w
formal demonstration of S protein cleavage upon entry is cur- stomatitisvirusGprotein-pseudotypedMLVusingthepQCXIN-CCM n
rentlylacking.Inthepresentstudy,wedevelopedanovelunbiased packaging vector. The polyclonal HEK-CCM, HeLa-CCM, and Vero- lo
a
approachtoselectivelyidentifyandcharacterizetheSproteinsof CCMcelllinesstablyexpressingCCM,aswellasmurineLR7cells,were d
e
incoming viruses that accomplish fusion. The assay employs a selected and maintained with G418 (PAA). CCM expression was con- d
combinationofaproteinbiotinligase(BirA)andabiotinacceptor firmedbyimmunodetectionusingmousemonoclonalanti-CCManti- f
r
peptide added as an extension to the cytoplasmic tail of the S body(MAbCC1,providedbyK.Holmes[31]).PolyclonalLR7cellsstably om
protein. When incoming viral proteins gain access to the cyto- expressingbiotinproteinligase(LR7-BirA)weresimilarlymadewiththe
h
plasmofcellsexpressingBirAligase,theyarespecificallylabeled MLV-pseudotypedvirususingthepQCXIP-BirApackagingvector.LR7- tt
BirAcellswereselectedat15(cid:3)g/mlandmaintainedat10(cid:3)gofpuromycin p
withbiotin,whichthenenablesisolation,enrichment,anddetec- :
/
tion. With this assay, we investigated the S glycoprotein of the (Sigma,catalogno.P8833)/ml.BirAexpressionwasconfirmedbyimmu- /jv
prototypecoronavirusMHV(strainA59).TheMHVSproteins nodetection using Cy3-conjugated mouse monoclonal anti-FLAG i.a
(Sigma, catalog no. A9594). No BirA enzyme was detected in the cell s
arepartiallycleavedintothenoncovalentlylinkedsubunitsatthe m
culturesupernatantsofLR7-BirAcellsafter72hofincubation,asana-
S1/S2junctionbyfurinorfurin-likeproteases(20).Intriguingly, lyzedbyWesternblottingwithamousemonoclonalanti-FLAGantibody .o
preventingfurincleavagebymutationortheuseoffurininhibi- conjugatedtoHRP. rg
/
torshasnoeffectonvirusinfectivityofMHV(21–23).Usingour Conditionalbiotinylationassay.LR7orLR7-BirAcellswerecultured o
newapproach,wedemonstratethattheMHVSproteinspartici- toconfluenceinsix-wellclusters.Cellswereinoculatedwithvirus-con- n
A
patinginfusionareproteolyticallyprocessedinthetargetcellsata tainingcellculturesupernatantsupplementedwith50(cid:3)gofDEAE-dex- u
differentpositionintheS2subunit.ThenewlyidentifiedS2*sub- tran(Sigma,catalogno.D9885)/mland10(cid:3)Mbiotin(Sigma,catalogno. g
u
unithascharacteristicsofthefunctionalfusionmachinery. B4639)atamultiplicityofinfection(MOI)of10.After30min,protein s
biosynthesis was inhibited by the addition of 50 (cid:3)g of cycloheximide t 1
1
(Sigma,catalogno.C7698)/mltopreventSproteinsynthesisfromvirus
MATERIALSANDMETHODS ,
infections.At90minpostinfection(p.i.),thecellswerechilledonice, 2
Cells,viruses,antibodies,andHR2peptide.HEK-293T,HeLa,Vero- washedtwicewithice-coldphosphate-bufferedsaline(PBS),andlysedin 01
CCL81,andLR7(24)cellsweremaintainedinDulbeccomodifiedEagle ice-coldradioimmunoprecipitationassay(RIPA)buffer(150mMNaCl, 5
mediumsupplementedwith10%fetalbovineserum.Generally,MHV b
1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl y
(strainA59)waspropagatedandtitratedinLR7cellsinculturemedium
sulfate[SDS],50mMTris-HCl[pH8])supplementedwithComplete U
supplementedwith20mMHEPES.FortheimmunedetectionofSpro- proteaseinhibitorcocktailtablets(Roche,catalogno.11836153001)to n
teininvirussupernatants,MHVwasgrowntohightitersinDulbecco iv
preventfurtherproteolysisandwithorwithout6mMsodiumpyrophos-
modified Eagle medium supplemented with 0.3% tryptose phosphate o
phate(PP;Sigma,catalogno.71516)toquenchtheactivityofBirAincell f
broth(Sigma,catalogno.T9157).Forimmunoprecipitation(IP)andim- lysates(32).Thecelllysateswereclearedbycentrifugationat10,000(cid:4)g M
mBAuPneadnetitbecotdioyn(,GMenHSVcrSippt,roctaetianlowgasnroe.aAct0e0d6w74it)hoprolmycolounsealmraobnboitclaonntai-l for10minat4°C.Thesupernatantswerecombinedwith20(cid:3)lof50:50 iss
slurryofproteinG-Sepharose(Biovision,catalogno.6511)supplemented o
anti-S2(10G)antibodyandsubsequentlywithanti-mouseoranti-rabbit u
with 0.5 mg of polyclonal anti-BAP antibody (GenScript, catalog no. r
immunoglobulinGconjugatedtohorseradishperoxidase(HRP;Dako, i
A00674)andincubatedunderrotationfor2hat8°Ctoimmunoprecipi- A
cwaatsaluosgednoto.Pd0et2e1c7t)in(2fe5c,t2ed6)c.eAllspboylyrcelaocntiaolnrawbibthitaannttii--rMabHbVitismermumun(oKg1lo3b5)- tatetheSproteins.Next,Sepharosebeadswerepelletedat6,000(cid:4)gfor5 t S
ulinGconjugatedtoHRP.Biotinwasdetectedbystreptavidin-HRPcon- minat4°Candwashedtricewithanexcessofice-coldRIPAbuffer.Excess t L
jugate(ThermoScientific,catalogno.21126).TheMHVfusioninhibitor supernatantwascarefullyremoved,andsampleswerefinallydenaturedby o
u
HR2 peptide (DLSLDFEKLNVTLLDLTYEMNRIQDAIKKLNESYIN theadditionofsamplebufferandsubjectedtoWesternblotting. is
Ifinhibitorycompoundswereusedduringtheinfection,cellswere
LKE)wassynthesizedbyGenScript(11).
Construction of recombinant viruses. Recombinant MHVs were pretreatedfor30minat37°C,followedbyinfectioninthepresenceofthe
generatedbytargetedrecombinationasdescribedearlier(27).Atransfer respective compounds. The following protease inhibitors were used at
vector based on pXHERLM was generated to create the recombinant theirhighestrecommendedworkingrangeconcentrationsaccordingto
MHV-BAPvirusencodingatandemrepeatofthe15-amino-acidbiotin Sigma’s protease inhibitor technical bulletin INHIB1 (final concentra-
acceptorpeptide,includinglinkersDLPGGLNDIFEAQKIEWHEPPGGL tion):pepstatinA(1.5(cid:3)M;Sigma,P5318),leupeptin(100(cid:3)M,Sigma,
NDIFEAQKIEWHE(theBAPsequenceisunderlined)asaC-terminal catalogno.L2023),E64d(10(cid:3)M;Sigma,catalogno.E8640),phosphor-
extensionoftheSprotein(28).TherecombinantvirusesMHVFCS(cid:2)BAP amidon(10(cid:3)M;Sigma,catalogno.R7385),andAEBSF[4-(2-amino-
and MHVS2*(cid:2)BAP were generated by introducing additional point ethyl)-benzenesulfonylfluoride;100(cid:3)M;Sigma,catalogno.A8456].HR2
mutations into the transfer vector using site-directed mutagenesis. peptidewasusedat25(cid:3)M.Thefollowinglysosomotropicagentswere
MHVFCS(cid:2)BAPSproteincarriesthreepointmutations—R713S,R7174, used(finalconcentration):ammoniumchloride(25mMNH Cl;Merck,
4
andR717S—thatsubstituteallargininesatthefurincleavagesitebyser- Darmstadt,Germany)andbafilomycinA1(125(cid:3)M;EnzoLifeSciences).
4944 jvi.asm.org JournalofVirology
ProteolyticProcessingofMurineCoronavirusSProtein
Timecoursebiotinylationassay.LR7orLR7-BirAcellswerecultured cellanddelivertheirgenomeintothecytoplasm.Accordingtothe
toconfluenceon10-cmdishes,andtheinoculumwaspreparedsimilarto currentmodelofclassIproteinfusion,theC-terminaltailofthe
theconditionalbiotinylationassay.First,cellswerewashedtwicewith CoVSproteinishiddeninternallyintheintactvirion.Itwillbe
ice-coldPBS,andice-coldinoculumwasaddedfor45mintoallowat- introducedintothecytoplasmafterthevirusandcellmembrane
tachmentofthevirustothetargetcellsat8°C.Next,theinoculumwas
havefused.InordertobeabletodiscriminateSproteinscoming
removed,andthecelllayerwaswashedoncewithice-coldPBS,followed
from virions that successfully achieved fusion from those that
bytheadditionof37°Cculturemediumsupplementedwith10(cid:3)Mbiotin.
failed,wedesignedabiotinylationassaythatusesselectiveintra-
Differentialperiodsofinfectionwereachievedbysuccessivelydelaying
cellularbiotinlabelingoftheprotein’sCterminus.Tothatend,we
thestartofattachmentandinfectionwhilemaintaininganequalduration.
generatedarecombinantMHV-A59derivativecarryinganSpro-
Allsampleswereharvestedatthesametimetoevenoutthetimebetween
lysis and immunoprecipitation. Then, 50 (cid:3)g of cycloheximide/ml was teinwithaC-terminallyappended37-amino-acidbiotinacceptor
added30minafterwarminguptheinfectionto37°Cforallsampleswith peptide(MHV-BAP;Fig.1A)andarecombinantmurinecellline D
aninfectionperiodlongerthan30minorattheendoftheinfection.The that constitutively expresses BirA in its cytoplasm (LR7-BirA). o
w
samplefor0minwaspreparedforlysisafter1minat37°C.Lysisand BirArecognizesthebiotinacceptorpeptide(BAP)asthesubstrate
n
immunoprecipitationwasperformedasdescribedintheconditionalbi- forbiotinligationinthepresenceofATPandfreebiotin.Inintact lo
otinylationassay,andIPsampleswereanalyzedbyWesternblotting. virions,theBAPfacestheluminalsideandisprotectedfrommod- ad
HR2 inhibition of MHV infection. Multiple wells containing LR7 ificationbyBirA,butuponvirus-cellfusionitbecomesexposedto e
cellswereinfectedwithwild-typeMHVfor1.5mintosynchronizeinfec- d
theenzyme(Fig.1B).Consequently,BirAcanbiotinylatetheBAP f
tion.Theinoculumwasreplacedbyculturemediumatthestartofinfec- r
tagofSproteinsofvirionsthatunderwentfusion,enablingthe o
tion.Atincreasingtimepoints,supernatantsofindividualwellswerere- m
plenishedwithculturemediumsupplementedwith20(cid:3)MHR2peptide selectionandcharacterizationofpostfusionSproteinsviathebi- h
toblockMHVentry.At4hp.i.,thesupernatantwasreplacedwithculture otin label. MHV-BAP displayed similar growth kinetics but tt
p
mediumcontaining1(cid:3)MHR2peptidetoinhibitsyncytiumformation. yielded10-fold-reducedtiterscomparedtowild-typeMHV(data :/
/
At7hp.i.,thecellswerefixedwith3.7%formalin,immunoperoxidase notshown). jv
stainingwasperformedusingK135serum,andthecellswerevisualized To characterize the S protein of MHV-BAP and to demon- i.a
usinganAECsubstratekit(VectorLaboratories).Theextentofinfection stratebiotinylationoftheBAPtag,wepropagatedwild-typeMHV sm
relativetononinhibitedvirusinfectionwascalculatedfromthenumberof andtherecombinantMHV-BAPinLR7cellsandLR7-BirAcells. .
o
plaquesobserved. ThecellculturesupernatantswereanalyzedbyWesternblotting rg
Deglycosylation.LR7orLR7-BirAcellswereculturedtoconfluence withantibodiesrecognizingtheS2subunitortheBAPtag,orwith /
in10-cmdishes,andthevirusinfectionwasperformedsimilarlytothatof o
thebiotin-bindingstreptavidin(Fig.1C).Themonoclonalanti- n
theconditionalbiotinylationassay.AfterIP,samplesontheSepharose
A
bodyrecognizingtheS2subunitdetectedthefull-lengthSprotein
beadsweredenaturedanddeglycosylatedwithPNGaseF(NewEngland u
BioLabs,catalogno.P0704)accordingtothemanufacturer’sprotocol. (S0) and the S2 subunit of all virus preparations. A polyclonal gu
Finally,thesamplesweredenaturedbytheadditionofsamplebufferand antibodydirectedagainsttheBAPspecificallydetectedS andthe s
0 t
subsequentlyanalyzedbyWesternblotting. S2subunitofMHV-BAPbutnotthoseofwild-typeMHV.Im- 1
1
Westernblotanalysis.ForthedetectionofSproteininvirus-contain- portantly,biotinylationofBAP-taggedSproteinwasonlydetected ,

ingcellculturesupernatants,aliquotsweredirectlylysedanddenaturedin forMHV-BAPvirusesproducedinLR7-BirAcells,demonstrating 2
0
samplebuffercontaining50mMTris-HCl(pH6.8),50%glycerol,5% theBirA-dependentbiotinylationoftheBAP.RecognitionoftheBAP 1
2-mercaptoethanol,1%SDS,andbromophenolblueandboiledat95°C 5
bytheanti-BAPpolyclonalantibodywasnotinfluencedbyitsbioti- b
for10min.Samplesafterimmunoprecipitationwereelutedfrombeadsby nylation status; tagged and nontagged S proteins were detected y
boilingat95°Cfor10mininsamplebuffer.Supernatantwassubjectedto U
equallyefficient.
SDS-PAGEinadiscontinuousgelwith8%acrylamideintheseparating n
gel (33). Next, samples were transferred to a polyvinylidene fluoride BiotinylatedSproteinsaftervirus-cellfusion:detectionof iv
membrane(Bio-Rad,catalogno.162-0176).Membraneswereblocked S2*.Next,weassessedthebiotinylationofSproteinsaftermem- of

withbovineserumandreactedwithantibodiesorstreptavidin-HRPin branefusionofvirionswithBirAexpressingtargetcells.TheLR7- M
PBSwithbovineserumand0.5%Tween20.Fordetection,weusedan BirAcellswereinoculatedwithMHV-BAP(MOI(cid:5)10)for90min is
s
AmershamECLWesternblotanalysissystem(GEHealthcare,catalogno. to enable binding and fusion. To detect the biotinylated S pro- o
u
RPN2109)withX-OmatLSfilms(Kodak;Sigma,catalogno.F1149). teins, anti-BAP antibody was used to immunoprecipitate the r
Computationalanalysis.ThetransmembranedomainofMHVSpro- BAP-taggedSproteinsfromwhole-celllysates.Thispurification i A
teinwaspredictedbyTMHMM2.0,andthesignalpeptidewaspredicted andconcentrationstepwasessentialfordetection.TheBirAen- t S
by SignalP 4.1. HR1 and HR2 regions were defined according to the zymemaintainsitsactivityinthelysisbuffer,evenatlowtemper- t
methodofBoschetal.(11).GlycosylationsiteswerepredictedwithNet- L
atures. Consequently, all S protein present in the cell lysate be- o
NGlyc1.0(TechnicalUniversityofDenmark).Westernblotsignalswere u
quantified by using ImageJ. Amino acid sequence alignment was per- came biotinylated postlysis and could be detected using the is
streptavidin-HRPconjugate(Fig.2A,lane1).InadditiontotheS
formedbyCLUSTALW2usingSsequencesofinfectiousbronchitisvirus 0
(IBVstrainBeaudette,NP_040831.1),MiddleEastrespiratorysyndrome andS2forms,whichcouldalreadybeobservedinthevirusstock
coronavirus(MERS-CoV;strainHCoV-EMC,AFS88936.1),mousehep- (Fig.1C),anadditionalproductof(cid:6)80kDawasdetectedwhich
atitis virus (MHV strain 2, AAF19386.1 and strain MHV-A59, wenamedS2*.Topreventpostlysisbiotinylationandanalyzethe
NP_045300.1), severe acute respiratory syndrome coronavirus (SARS- S proteins as they occur in the intact cell, BirA activity was
CoVstrainTor2,NP_828851.1),andtransmissiblegastroenteritisvirus quenchedbyproductfeedbackinhibitionbytheadditionofPPto
(TGEVstrainTO14,AF302263_1). thelysisbufferandduringtheIPprocedure(Fig.2A,lane2)(32).
TheS2*wasthemostabundantSproteinproductdetected,and
RESULTS onlylimitedamountsofS andS2wereobserved.
0
Biotinylation assay to label S protein after virus-cell fusion. TotestwhethertheappearanceoftheS2*proteinindeedcor-
Duringinoculation,notallvirionssuccessfullyfusewiththetarget relateswithsuccessfulinfection,weexploitedtheHR2peptide,a
May2014 Volume88 Number9 jvi.asm.org 4945
Wichtetal.
A S1/S2 junctionS2’
MHV S protein
717 869
SP HR1 HR2
ΨΨ Ψ Ψ Ψ Ψ ΨΨΨΨ ΨΨ Ψ ΨΨΨ
N- -C
FP TM
MHV S protein with carboxy-terminal BAP BAP
1324-1361
N- -C
S
S1 0 S2
B D
biotin o
w
n
lo
a
d
e
FIG2DetectionofSproteinaftermembranefusionwithtargetcells.(A) d

LR7-BirAcellswereinoculatedwithMHV-BAPfor90minintheabsenceor fr
CMV BirA presenceofpeptidicfusioninhibitor(HR2)andlysedintheabsenceorpres- om
enceoftheBirAinhibitorPP.ImmunoprecipitatedSproteinswereanalyzed
h
byWesternblotting.OnlybiotinylatedSproteinwasdetectedbystreptavidin- t
t
HRPconjugate.UponcelllysisintheabsenceofPP,allSproteinwasallowed p
:
C ttohebSe1b/iSo2tijnuynlactteiodnb(ySB2i)r,Aa;ntdhuasn,fouvlel-llepnrogtdhuSctporoftloeiwne(rSm0),oSlepcruolaterimncalsesadveedsiga-t //jv
virus: MHV MHV-BAP MHV MHV-BAP MHV MHV-BAP natedS2*weredetected.ThepresenceofPPduringlysisallowedtheexclusive i.a
cells: LR7 LR7-BirLAR7 LR7-BirA LR7 LR7-BirLAR7 LR7-BirA LR7 LR7-BirLAR7 LR7-BirA dtheeteSc2ti*ofnraogfmSepnrto.t(eBin)ItPhastawmapslbesiowtienryeldateendatduurreidngatin9f5ecotrio6n5°aCndbemfoarienWlysehstoewrns sm.
o
blotting.InthepresenceofPP,theS2*fragmentconstitutesthemajorityof
r
170- -S0 biotinylatedSproteinsmigratingat(cid:6)80kDaposition.AfterheatingIPsam- g/
130- plesat65°C(insteadof95°C),alargerproductwaspresentaroundthe(cid:6)200- o
-S2 kDaposition(indicatedbytheasterisk). n
100- A
u
70- g
55- thatbiotinylationofSproteinsonlyoccursaftervirus-cellfusion us
WB:MHV-S2 WB:BAP WB:biotin andfurtherdemonstratedthattheproteolyticprocessingofSpro- t 1
1
FIG1RecombinantMHV-BAPforthedetectionofbiotinylatedSproteins. teinisnotaffectedbytheadditionoftheHR2peptide(Fig.2A, ,

(A)Schematicorganizationofwild-type(1,324aminoacids)andBAP-tagged lane3).WehypothesizedthattheS2*subunitrepresentsthepro- 2
0
MHV(strainA59)Sprotein(1,361aminoacids)drawntoscale.TheSprotein teolyticallyprimedsubunitofMHVSprotein. 1
hasanN-terminalS1domain,responsibleforreceptorbinding,andaC-ter- 5
minalS2domainthatholdsthefusionmachinery.Thepositionsofthesignal TheS2*subunitoccursinstablemultimers.Weexaminedthe b
peptide(SP),thepredictedN-glycosylationsites((cid:7)),theputativefusionpep- novelS2*subunitforcharacteristicfeaturesofthefusionmachin- y
U
tide(FP),twoheptadrepeat(HR)regions,thetransmembranedomain(TM), ery. To drive membrane fusion, the membrane-anchored do- n
andthebiotinylationacceptorpeptide(BAP)areindicated.MHVSprotein mainsofclassIfusionproteinsfoldintoahighlySDS-stableand iv
containstwopredictedproteasecleavagesites:afurincleavagesiteattheS1/S2 temperature-resistantpostfusiontrimer,facilitatedbythezipping o
junctionandaputativesecondcleavagesite(S2=)justupstreamofthefusion f
upofthetwoHRdomainsintosix-helixbundles(11,34).Totest M
peptide.(B)Abiotinylationassaywasdesignedtoselectivelyidentifyandchar-
acterizetheSproteinofMHVvirionsthatunderwentmembranefusionwith whether S2* forms such stable trimers, we analyzed the SDS- is
s
thecellulartargetmembrane.ThebiotinacceptorpeptidewasfusedtotheC PAGEmigrationofthebiotinylatedSproteinvariantsafterheat o
terminusoftheSprotein.Targetcellsstablyexpresscytoplasmicproteinbiotin treatment of the samples at 65°C rather than at 95°C. The S2* ur
ligase(BirA)fromacytomegaloviruspromoter.Uponmembranefusion,the i
subunit that was biotinylated upon infection of target cells mi- A
Cac-cteesrsmedinaalnBdAbPiootifnSylaptreodteibnyisBiirnAtr.o(dCu)ceCdhianrtaocttehreizacytitoonplaosfmreacnodmcbainnabnet gratedat(cid:6)80kDaifdenaturedat95°C.Thisspecieswas,however, t S
MHV-BAP.VirusstocksofMHV-A59withwild-typeSprotein(MHV)or absentafterdenaturationat65°C;instead,alargerbandwasob- t
L
withBAP-taggedSprotein(MHV-BAP)wereproducedinLR7cellsorLR7 servedat(cid:6)200kDa,inlinewiththeS2*subunitactuallyoccur- o
cinegllsaeMxpHreVss-iSn2gmBiorAno(cLloRn7a-BlairnAti)baonddy,suanbjeacntteid-BtAoPWpeostlyecrnlobnlaoltatinntgib(oWdyB,)aunsd- ringasastable,multimericpostfusioncomplex(Fig.2B). uis
ThekineticsofS2*appearance,viruscellfusion,andMHV
streptavidin-HRPforthedetectionofproteins.Full-lengthSprotein(S)and
S2subunitresultingfromcleavageattheS1/S2junctionweredetected0with infectioncoincide.IfthenovelS2*subunitrepresentstheproteo-
antibodiesdirectedagainsttheS2domainorBAPandwithstreptavidinforthe lyticallyprimedform,thekineticsofSproteincleavageshouldbe
detectionofbiotinylation.Thesizesandpositionsofmarkerproteins(inkilo- equaltoorfasterthanproductiveMHVinfection.Wemonitored
dalton)areindicatedontheleft.
thekineticsofSproteincleavageandfusionbyperformingatime
course of infection with MHV-BAP on LR7-BirA cells. To syn-
chronizeinfections,viruswasallowedtobindtocellsat8°Cfor1
synthetic peptide fusion inhibitor, which effectively blocks the h,followedbyremovaloftheinoculum,afterwhichinfectionwas
membranefusionactivityoftheSprotein(11).Additionofthe continuedat37°C.OmittingPPduringtheIPprocedurerevealed
HR2peptideefficientlyabrogatedbiotinylationoftheSproteinin the overall biochemical fate of all (i.e., fused and nonfused) S
thepresenceofPP(Fig.2A,lane4).Theexperimentconfirmed proteinsovera90-mintimeperiod(Fig.3A).Westernblotanal-
4946 jvi.asm.org JournalofVirology
ProteolyticProcessingofMurineCoronavirusSProtein
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
FIG3S2*fragmentisgeneratedduringvirusentry.(A)MHV-BAPwasboundtoLR7-BirAcells,andexcessviruswasremovedtosynchronizetheinfection.The jv
infectionwasstoppedattheindicatedtimespostinfectionbycelllysisintheabsenceoftheBirAinhibitorPP.ImmunoprecipitatedSproteinswereanalyzedby i.a
Westernblotting,andbiotinylatedproteinwasdetectedbyusingastreptavidin-HRPconjugate.Toassesslysosomaldegradation,ammoniumchloride(NHCl) s
4 m
wasaddedduringinfection.Abackgroundband(bg)isindicated.TherelativeamountsofS,S2,andS2*proteinperlanewerequantifiedtoillustratethe
proteolyticprocessingovertime(lowerpanel).(B)SameaspanelA,exceptthatbyadditionof0PPduringlysis,onlySproteinthathasbeenbiotinylatedduring .o
r
infectionwasdetected.Ascontrols,MHV-BAPintheabsenceofcells,infectionwithwild-typeMHV,andMHV-BAPinfectionperformedinthepresenceofthe g
/
HR2fusioninhibitorwereused.TheintensityoftheS2*fragmentwasquantifiedandisdisplayedasabardiagrambelow.Inaddition,todeterminethekinetics o
ofvirusentryindependently,MHVinfectionsweresupplementedintimewiththeHR2fusioninhibitorafterasynchronizedinfection(linediagram).At7hp.i., n

infectedcellsweredetectedbyimmunostaining,andtherelativeamountsofinfectionweredetermined. A
u
g
u
s
t
ysis of IP samples indicated that the relative amount of the S2* yieldingtheS2*productbutwasslower.MHVinfectioncoincided 1
1
cleavageproductincreasedovertime,whereastheS0andS2signal withtheaccumulationoftheS2*subunitasmonitoredbyintra- ,
slowlyvanished.Topreventthematurationofendosomesandthe cellularbiotinylation.Incomparison,theintensitiesofthebiotin- 2
0
acidificationofendolysosomalcompartments,25mMNH Clwas ylatedS2*proteinbandsobservedinthevirus-cellfusionexperi- 1
4 5
addedtothecellsthroughoutinfection(35).Thistreatmentre- ment were quantified and included in the same graph as a bar b
sulted in a net increase of S protein, suggesting that the time- chart(Fig.3B,barchart). y

U
dependentoveralldecreaseinSproteinintheabsenceofNH Cl ConservedarginineisnotthecleavagesitethatyieldstheS2*
4 n
was due to lysosomal degradation. In contrast, the proteolytic subunit. The identification of the proteolytic cleavage site that iv

process leading to S2* formation was not blocked by NH Cl. yieldsthefusionactiveS2*subunit,couldprovidefurtherinfor- o
4 f

QuantificationofthedensityoftheSproteinbandsover90minof mation about the requirements for gaining fusion competence. M
infection showed that the fraction of the S2* subunit increased JudgedfromthemolecularweightoftheS2*protein,thecleavage is
s
from3to50%(Fig.3A,barchart).However,notallSproteinshad siteislocatedwithintheN-terminalhalfoftheS2subunit.This o
u
undergoneproteolyticprocessingafter90min.Byperformingthe regioncomprisesaconservedarginine,previouslydescribedasa r
analysisinthepresenceofPPduringthesamplepreparation,only potentialproteasetargetsiteandtermedS2=intheSproteinsof i A
t
Sproteinfromvirus-cellfusioneventswasmonitored(Fig.3B). SARS-CoVandIBV(18,36)(Fig.4A).Cleavageatthisarginine S
The appearance of the S2* subunit started early and continued wouldtruncatetheS2domainby(cid:6)15kDaandremovetwogly- t
L
increasinglyforatleast90min. cosylationsites,whichisinagreementwiththeobserveddiffer- o
u
Toconfirmthekineticsofvirus-cellfusionbyanindependent ence between the S2 and S2* band. We used a reverse-genetics is
approach,weexaminedtheinhibitionofMHVinfectionbyHR2 approachtodeterminewhethertheMHVS2*subunitindeedre-
peptidefusioninhibitor.LR7cellswerepulseinoculatedfor1.5 sultsfromproteolysisattheputativeS2=cleavagesite.Tothatend,
minwithwild-typeMHVtosynchronizebinding.Inoculawere mutantMHV-BAPwasgeneratedcontainingtwoserinesubstitu-
replacedbyculturemedium,afterwhichHR2peptidewasadded tions of arginines occurring at or close to the S2= cleavage
to individual samples at successive time points. The relative (MHVS2=(cid:2)BAP;Fig.4A,table).AnotherMHVvariantwithamu-
amountofinfectionwasdeterminedat7hp.i.byimmunestain- tated furin cleavage site at the S1/S2 junction was generated by
ingofthecellsagainstMHV.ThepresenceofHR2peptidefrom replacing the arginines by serines (MHVFCS(cid:2)BAP). Mutant vi-
the start completely abolished infection but showed no effect ruseswereviableandusedtoinfectLR7-BirAcellsfor90minatan
whenadded120minafterinoculation(Fig.3B,linechart).The equalMOI,afterwhichIPsampleswerepreparedintheabsence
MHVinfectiondeducedfromtheHR2peptidetime-of-addition orpresenceofPP.WesternblotanalysisofIPsamplesshowedthat
experiment showed similar kinetics to proteolysis of S protein the knockout of the furin cleavage site at the S1/S2 junction in
May2014 Volume88 Number9 jvi.asm.org 4947
Wichtetal.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
jv
i.
a
s
m
.
o
r
g
/

o
n

A
u
g
u
s
t

1
1
,

2
0
1
5

b
FIG4CharacterizationoftheS2*fragment.(A)SequencealignmentofanSproteinsegmentofrepresentativeCoVscontainingtheputativeS2=cleavagesite y

(arrow)andpartofthefusionpeptide(FP).Theconservedarginineresidueadjacenttotheputativefusionpeptide(FP[37])isindicatedinboldface.Thetable U
showsthemutationsofgeneratedrecombinantMHVvirusescarryingserinesubstitutionsofarginines(underlined)atthefurincleavagesiteattheS1/S2junction n
(MHVFCS-BAP)andtheputativeS2=site(MHVS2=-BAP).Theaveragetiterofthreeindependentviruspreparationswasdeterminedbyendpointdilutionandis iv

reportedasthe50%tissuecultureinfectivedose(TCID )/ml.BirAcells,infectedwiththerecombinantvirusesfor90min,werelysedintheabsenceorpresence o
ofpyrophosphate(PP).Biotinylationofimmunoprec5ip0itatedSproteinswasdetectedbyWesternblotting,asdescribedinthetext.(B)Deglycosylationof f M
biotinylatedSproteins.ThebiotinylationassaywasperformedwithMHV-BAPasdescribedforpanelA.PriortoWesternblotanalysis,allsampleswere is
denatured,andselectedsamplesweresubsequentlydeglycosylatedusingPNGaseF.BiotinylatedSproteinwasdetectedwithstreptavidin(twoexposuretimesare s
o
shown),andthesameblotwasrestainedwithanti-BAPantibodytodetect(nonbiotinylated)Sproteins(notethatstreptavidinbindingtobiotinylatedBAPlimits u
detectionwiththeanti-BAPantibody).Full-lengthSproteinisindicatedbyopentriangles,SproteincleavedattheS1/S2junctionisindicatedbysolidtriangles, r
i
andtheS2*fragmentisindicatedbyasterisks.AllSproteinsshowedfastermigrationafterdeglycosylation;S andS2werereducedtoadefinedbandupon A
0
deglycosylation,whereastheS2*fragmentbandremaineddiffuse.Acellularbackgroundbandisindicated(bg). t
S
t

L
MHVFCS-BAPpreventedtheappearanceoftheS2form(22)(Fig. PredictionoftheS2=cleavagesitefromthemolecularweight o
u
4A,lowerpanel).Incontrast,serinesubstitutionofthetwoargi- oftheS2*subunitafterdeglycosylation.Sincewecouldnotpre- is
ninesatthepresumedS2=cleavagesiteinMHVS2=-BAPdidnot dictotherproteasecleavagesitesfromtheSproteinaminoacid
preventtheformationoftheS2*subunit.WhenIPwasperformed sequence,wetriedtoidentifytheS2=cleavagesitebyalternative
inthepresenceofPP,onlyallowingthedetectionoftheSproteins approaches. The biotinylation assay did not yield a sufficient
involved in fusion, the S2* subunit was clearly detected for all amountandpurityoftheS2*subunittoallowN-terminalamino
three viruses. The S2* product of the mutant viruses migrated acidsequencing.Instead,wedeglycosylatedtheSproteintomore
withsimilarmobilityandrepresentedthemajorformofSprotein preciselydeterminethemolecularweightofS2*,fromwhichthe
thatunderwentfusion.ArgininesubstitutionsattheS1/S2orS2= approximatelocationofthecleavagesitemightthenbededuced.
sitehadnodetectableeffectonvirustiters,whichremainedcom- Tothatend,LR7-BirAcellswereinoculatedwithMHV-BAPfor
parabletoMHV-BAP(Fig.4A).Asreportedearlier,thedeletion 90min,andsampleswerepreparedintheabsenceorpresenceof
oftheS1/S2argininemotifresultedinreducedsyncytiumforma- PP.AftertheIP,SproteinboundtoproteinG-Sepharosebeads
tioncapacityofthevirus(22). wasdenatured,andsamplesweredeglycosylatedbyPNGaseFto
4948 jvi.asm.org JournalofVirology
ProteolyticProcessingofMurineCoronavirusSProtein
remove all N-linked glycans. Successful deglycosylation was re-
vealed by the S proteins migrating with higher electrophoretic
mobility(Fig.4B,topandmiddlepanel).Deglycosylationofall
Sproteins((cid:2)PP)andofSproteinsfromvirionsthathadfused
((cid:8)PP) showed a similar effect. The theoretical molecular
masseswerepredictedtobe70kDafortheS2domainand54
kDaforS2*ifthecleavageoccursclosetotheputativeFP.The
deglycosylatedS2subunitshiftedfromthe105-kDapositionto
the80-kDaposition—slightlyhigherthanpredicted—andmi-
gratedasawell-definedband.Incontrast,theS2*proteinalso
shiftedtoalowermolecularmass,andyetitremainedhetero- D
o
geneous after deglycosylation, ranging in mass from 60 to 65 w
kDa.SimilartoS2,theS2*subunitappearslargerthanitspre- n
lo
dictedmolecularmassof54kDa;hence,cleavagemayoccurat a
theputativeS2=cleavagesiteorfurtherupstream.Theblotwas de
restainedwithantiserumagainsttheBAPinordertovisualize d

f
theSproteinsfrominfectionsofLR7cellwithoutBirA(Fig.4B, ro
m
lower panel) and independent of biotinylation. Of note, the

priorstreptavidin-biotininteractionreducestheanti-BAPan- h
t
t
tibodyreactivity,particularlyinfullybiotinylatedsamplespre- p
:
/
paredintheabsenceofPP(Fig.4B,lanes2and3). /jv
Inhibition of cellular proteases that generate the S2* sub- i.
a
unit.ToobtaininformationontheSproteincleavagesiteandthe s
m
functional aspect of proteolysis during virus infection, we at- .
o
temptedtoidentifytheresponsiblehostcellproteases.SARS-CoV r
g
Sproteincanbecleavedbymultipleproteases,andtheavailability /
o
ofthoseproteaseshasbeenlinkedtothetissuetropismofthevirus n

(7). However, expression of the MHV receptor can render cell A
u
linesofdifferentspeciessusceptibletoMHVinfection(31),andif g
u
the S2* subunit represents the fusion active form, the priming s
t
protease(s)shouldoccurinvariouscelllines.Totestthis,wemon- 1
1
itoredSproteincleavageinnonmurinecelllinesstablyexpressing ,

theMHVreceptor.Viruspreparationscontainingprebiotinylated FIG5ProteaseinhibitorsorlysosomotropicagentsdonotpreventS2*for- 20
mation.(A)BiotinylatedMHV-BAP(MHV-BAP*bio)progenyviruseswere 1
S protein were produced after a single passage on LR7-BirA producedinLR7-BirAcells.CellsoverexpressingtheMHVreceptormurine 5
(MHV-BAP*bio)andtypicallycontainedca.70%S0,30%S2,and carcinoembryonicantigen-relatedcelladhesionmolecule1a(CCM)werein- by
amarginalfractionofS2*(Fig.5A,lane1).After90minofinoc- fectedwithMHV-BAP*biofor90min.ImmunoprecipitatedSproteinswere
U
ulationoftwohumanandonesimiancellline(i.e.,HEK293T, analyzedbyWesternblotting,andbiotinylationwasdetectedbyusingthe n
HeLa,andVerocells,respectively),thepatternsofS ,S2,andS2* streptavidin-HRPconjugate.ThecleavagestatusofMHV-BAP*biopriorto iv
0 infectionwasvisualizedbyinoculatingLR7-BirAwithvirus-containingcell o
weresimilarcomparedtothatinthemurineLR7-BirAcellline culturesupernatantfor2h15minat4°Canddirectlysiswithoutwarming f
M
(Fig.5A).Broad-spectrumproteaseinhibitorscanaffectvarious (firstlane).(B)LR7-BirAtargetcellswerepretreatedwithvariousbroad-spec-
classesofproteases.Inordertocharacterizetheproteasesinvolved trumproteaseinhibitorsfor30min.InfectionwithMHV-BAPwasallowedin iss
thepresenceofproteaseinhibitorsfor90min,andsamplepreparationwas o
in S protein cleavage, a virus entry assay was performed in the subsequentlyperformedintheabsenceofPPasdescribedforpanelA.(C) u
r
presenceofvariousproteaseinhibitors,suppressingtheactivityof SameaspanelB,exceptthatinfectionswereperformedwithMHVorMHV- i
A
themainclassesofproteases.MHV-BAPinfectionwasperformed BAPinthepresenceofHR2fusioninhibitor,ammoniumchloride(NH4Cl),or t
bafilomycinA1(BafA).LysateswerepreparedintheabsenceorpresenceofPP. S
onLR7-BirAcellsfor90mininthepresenceofthecysteinepro-
t

teaseinhibitorE64d,themetalloproteaseinhibitorphosphoram- L
o
idon,theaspartylproteaseinhibitorpepstatinA,theserineand u
is
thiolproteaseinhibitorleupeptin,andtheserineproteaseinhibi- DISCUSSION
torAEBSF(Fig.5B),aswellastheserineproteaseinhibitorcamo-
WestudiedthecleavageoftheMHVSglycoproteinduringvirus
stator1(cid:4)-concentratedRochemini-cocktailinhibitor(datanot
entrybyanunbiasedapproachthatallowedustoisolatefusion
shown).Inaddition,theinvolvementoflow-pH-dependentpro- proteins of virions that accomplished virus-cell fusion, and we
teases was probed using the lysosomotropic agents NH4Cl and newlyidentifiedanS2*subunit.Itdisplayedfeaturesofthefusion
bafilomycinA1(Fig.5C).Noneoftheappliedagentscouldpre- machinery,suggestingthattheS2*subunitrepresentsthefusion-
venttheSproteincleavagethatresultsintheformationoftheS2* activepartoftheSprotein.Insupportofthis,themajorityofthe
subunit.ThelysosomotropicagentsNH ClandbafilomycinA1 postfusion S proteins were cleaved into the S2* protein and
4
abolishedfusionsimilartotheHR2peptidefusioninhibitor,as formedheat-andSDS-stablemultimersthatresemblethepostfu-
indicatedbythelackofbiotinylatedSwhenIPwasperformedin sionsix-helixbundle.Furthermore,thekineticsofappearanceof
thepresenceofPP(Fig.5C). thebiotinylatedS2*proteincoincidedwiththekineticsofvirus
May2014 Volume88 Number9 jvi.asm.org 4949
Wichtetal.
entry,asdeterminedbymonitoringthesensitivityofinfectionto S2*, perhaps via a short-lived intermediate S2 form that is not
theHR2peptidefusioninhibitor.ThesizeoftheS2*proteinin- detected.Withfewerprimingproteasesonthecellsurfacethanin
dicates cleavage to occur in the S2= region just upstream of the theendolysosomalcompartments,thismayexplainthecell-cell
putative FP. We could not determine the exact cleavage site by fusion inability of the MHV spikes lacking a functional furin
reversegenetics,andthelowmassamountsoftheS2*proteindid cleavagesite.
notallowitsidentificationbymassspectrometry.Deglycosylation WeobservedthatSproteincleavageuponMHVinfectionoc-
oftheS2*proteinresultedinaheterogeneousproduct,suggesting curred downstream of the S1/S2 junction and released an S2*
thatcleavagecanoccuratalternativesitesinproximitytotheS2= fragmentof(cid:6)80kDa,whichis(cid:6)25kDasmallerthantheS2sub-
site.Proteaseinhibitorsusedtoidentifytheproteaseresponsible unit.Assumingthismembrane-boundsubunittocarrythemem-
forSproteincleavagecouldnotpreventtheformationoftheS2* branefusionfunction,weprobedtheS2*subunitforcriteriaof
subunit.Althoughtheprecisedetailsofthecleavageprocessre- thefusionmachinery.Firstofall,theS2*subunitwasthemost
D
mainenigmatic,theappearanceandcharacteristicsoftheS2*sub- abundant S protein species observed after virus-cell fusion and o
w
unitsupporttheideathatitrepresentsthefusion-readysubunit. hencelikelytobeinvolvedinmembranefusion.Weassumethat
n
Previous investigations of CoV fusion protein cleavage have S andS2proteinsdecoratevirionswhichfailedtoreachthecel- lo
0 a
monitored the infectivity of viruses or viruslike particles in the lularcompartmentwheretheappropriatestimuliandproteolytic d
presenceofproteaseinhibitorsoraftergeneticallymodifyingthe activityoccurforSproteinactivation.However,alimitednumber e
d
fusionprotein(14,16,21,23,36,37).Manystudiesdemonstrate ofvirionsreachesthefusioncompartment,whereamajorityofS f
r
cleavageofSproteinsdisplayedonthecellsurfacebyrecombinant proteins are proteolytically processed and triggered for fusion. o
m
proteases,butonlyafewverifyproteolysisinviruspreparations Second,S2*occurredinheat-anddetergent-resistantmultimers
h
upon exposure to recombinant proteases and soluble receptor indicativeofthecharacteristicclassIpostfusionsix-helixbundle. tt
p
(38,39).ThesestudiesconvincinglycorrelatedcleavageofSpro- Similarly,treatmentofMHV-2virionswithsolublereceptor,fol- :
/
/
teinwithitsmembranefusioncapacitybutfailedtodemonstrate lowedbyproteasetreatment,revealedanequivalentpatternofS , jv
cleavageduringvirusentryoridentifyingthefusion-competent S2,andS2*(38).CathepsinLandtrypsincleavedtheSprotein0, i.a
s
subunit. In fact, the biochemical fate of viral glycoproteins on yieldinga71-kDafragmentthatappearedasastable,postfusion m
virionsthatareenteringhostcellsatphysiologicalMOIsisdiffi- form, similar to S2* (38). Proteolytic priming of the MHV-2 S .
o
cult to study. Given the limited amount of virus even at high proteinsuponvirusentrywasearlierimplicatedbystudieswith rg
MOIs,thesignificantfractionofnoninfectiousparticlesineachvirus inhibitorsofendolysosomalproteasesandlysosomotropicagents /
o
preparation,andtherelativelylownumberofSproteinspervirion, andbytrypsinbypassexperiments,buttheactualprocessingin n

A
thespecificdetectionofSproteinsonsuccessfullyfusingvirionsisa cellswasnotconfirmed(6).Inourstudy,proteolyticprocessingof
u
greatchallenge.Inthepresentstudy,weestablishedanovelbiochem- theSproteinandvirus-cellfusion,asmeasuredbyintracellular g
u
icalassaybasedontheconditionalbiotinylationofproteinstocon- biotinylationoftheSproteinandbyanindependentvirusinfec- s
t
centrateandpurifyMHVSproteinsinvolvedinfunctionalvirus-cell tionassay,occurredwithsimilarkinetics.TheHR2peptidefusion 1
1
fusionevents.Thisenablestheidentificationandcharacterizationof inhibitorpreventsvirusfromfusionbyinhibiting6-helixbundle ,

fusedSproteinsincombinationwithmoreclassicalexperimentsus- formation(11).Consistently,italsopreventedSproteinsofin- 2
0
ingsite-directedmutagenesisandproteaseinhibitors.Ourapproach comingvirionsfrombecomingbiotinylated,allowingustodis- 1
5
excludescontributionsofnonfusedvirionsandthusfocusesonfunc- criminatethesequentialorderofcleavageandfusion.Ifcleavageis b
tionalfusionevents.Sincetheinfectingvirionstakeaphysiological aprerequisitefortheSproteintomediatefusion,thenHR2must y

U
entryroute,wedonotrelyonmimickingthefusionprocessbythe takeeffectaftertheproteolyticevent,andHR2peptideindeeddid
n
additionofsolublereceptor,exogenousproteasetreatment,orpH not affect the cleavage of S protein. We argue, based on these iv

shock.Theassaycanbeadaptedtomonitorthebiochemicalfateof findings,thattheS2*fragmentfulfillsthecriteriaofthefunctional o
f

structuralvirioncomponentsofanyenvelopedornonenvelopedvi- fusionprotein. M
rusuponentry. The difference in molecular weight between the S2 and S2* is
TheentryofvariousCoVsissupportedbydistinctproteases subunitspredictsthesuspectedcleavagesitetomap(cid:6)230amino so
u
thatcanactattheplasmamembraneofthetargetcellorinthe acidsdownstreamoftheS1/S2junction.Furthermore,primingof r
i
endosomal compartments (10, 13). For MHV-A59, proteolytic theclassIfusionproteinsoftenoccursdirectlyNterminalofthe A
t
processingattheS1/S2junctionenablesefficientcell-cellfusion, FP,whichhasbeendescribedasaconservedsequenceofapolar S
resulting in syncytium formation (40), and mutagenesis of the aminoacidsintheCoVSprotein(16,36,37).Bothpredictions t
L
cleavage site limits the syncytium size (23, 41). However, as we pointtowardtwocriticalarginineresiduesintheMHVS2domain o
u
showedearlier(22)andconfirmedherebysubstitutingallargi- and, intriguingly, cleavage at the same position (S2=) has been is
ninesattheS1/S2junction,theS1/S2cleavageisdispensablefor implicatedtoenableSARS-CoVSproteinfusion(8,10).Byanal-
fusionactivityandvirusinfectivity.Thisissupportedbyobserva- ogy,wesuspectedtheS2=cleavagesitetobeusedinMHV-A59S
tionswithanaturalisolate,MHV-2(42),orwithacell-passaged protein,butaftermutagenesisofbotharginines,theinfectivityof
MHV/BHKisolate(43),whichbothlackagenuinefurincleavage MHV remained unaffected, and the S protein cleavage pattern
siteandhencecarryuncleavedSproteinsontheirvirions.Inour uponfusionremainedunaltered.
study,onlysmallamountsoftheS2subunitwerepresentonviri- Inanattempttodeducethecleavagesitefromitsmolecular
ons that had fused, suggesting that S2 is not the fusion-active weight,weenzymaticallyremovedtheN-linkedglycansoftheS2*
form.Nevertheless,cleavageofMHVSproteinattheS1/S2junc- glycoproteinandanalyzeditssize.AlthoughthedeglycosylatedS
0
tionmayprovideadditionalstructuralflexibilitytoincreasethe and S2 proteins were reduced to sharply defined products, S2*
accessibilityofacleavagesiteforpriming,assuggestedearlierfor remainedilldefined,migratingasaheterogeneousbandranging
SARS-CoV(36).ItispossiblethatSproteinsareprocessedinto from60to65kDa.AssumingthattheS2andS2*productunder-
4950 jvi.asm.org JournalofVirology
ProteolyticProcessingofMurineCoronavirusSProtein
wentsimilarposttranslationalmodifications,thevariablesizeof S proteins does not occur in the producer cell; cleavage in the
theS2*fragmentcanbebestexplainedbypromiscuousproteo- targetcellprovidesanextralevelofspatialandtemporalcontrolof
lyticcleavages,whereasS2isformedbycleavagepreciselyatthe virus fusion. Thus, MHV receptor-induced conformational
S1/S2junction.Heterogeneityofcleavageproductsmightresult changesareinitiatedatthetargetcell,exposingaproteolyticcleav-
from a certain degree of plasticity of S2= cleavage either by the agesite(19,38,47).SARSCoVSproteinsrequireafirstcleavageto
existenceofalternativecleavagesitesorbyinvolvementofmulti- facilitate a consecutive cleavage that then renders the S protein
ple or alternative proteolytic enzymes, analogous to the fusion fusioncompetent(36).Nevertheless,anadditionaltriggerofun-
activationoftheSARS-CoVSprotein(13).Theplasticityofthe knownnatureisprobablynecessarytoinitiatethemembranefu-
cleavagesitealsosuggeststhatcleavagedirectlyadjacenttotheFP sion,sincewecouldblockvirus-cellfusion,usinglysosomotropic
maynotbeanabsoluterequirementforfusion. agents,butnotSproteincleavage.LowpHintheendolysosomal
Inoursearchforthecleavagesite,weappliedbroad-spectrum compartmentmayitselfbeatriggerbutmayaswellbenecessary
D
protease inhibitors to identify corresponding (classes of) pro- forprimingbylow-pH-activatedproteases(14,48,49).Triggers o
w
teases.TestingproteaseinhibitorsinSARS-CoVentryhighlighted ofanalternativenatureseem,however,morelikelybecausethe
n
theinvolvementofcathepsinLandeventuallyledtotheidentifi- infectionofsomeCoVscanbebypassedusingrecombinantpro- lo
a
cationofthecathepsinLcleavagesiteintheSprotein(15,17).In teaseswithoutapHdecrease,whereassyncytiumformationtypi- d
contrasttoSARS-CoV,theproteaseinhibitorsleupeptinandE64d cally occurs at neutral pH (6, 20, 22, 39, 40). In summary, the e
d
and specific cathepsin L/B inhibitors failed to block MHV-A59 priming of S proteins plays a pivotal role in the temporal and f
r
infection(6,14).Weobservednoeffectontheproteolyticpro- spatialregulationofCoVentry.Withtheconditionalbiotinyla- o
m
cessing of MHV S proteins for broad-range protease inhibitors tionassaydescribedhere,theprimingeventsthatoccurafterre-
h
targeting cysteine, aspartyl, serine, thiol, and metallo proteases. ceptorbindinganddependoncellularproteasescanbecharacter- tt
p
WeconcludethatheterogeneityoftheS2*subunit,ourfailureto izedindetail. :
/
/
knockouttheS2=cleavagesitebymutation,andtheinsensitivityof jv
MHVtowardindividualproteaseinhibitorsareallconsequences ACKNOWLEDGMENTS i.a
s
ofredundantproteasesand/ormultiplecleavagesitesthatmediate WeacknowledgeZouYongforkindlyprovidingVeroCCL-81cells. m
MHVSproteinpriming.However,agivenproteaseinhibitormay ThisstudywassupportedbyEUFramework7programPITN-GA- .o
notblockallindividualproteasesofaspecificclass,andourinhib- 2009-235649-VirusEntry. rg
itorpanelwaslackingthreonineproteaseinhibitorsandamino- /
o
peptidase inhibitors that potentially prime the S protein (44). REFERENCES n

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