Rabies Virus Protein Synthesis in Infected BHK-21 Cells

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JOURNALOFVIROLOGY,Apr.1977,p.102-112Vol.22,No.1Copyright©1977AmericanSocietyforMicrobiologyPrintedinU.S.A.RabiesVirusProteinSynthesisinInfectedBHK-21CellsH.PAULMADORE*ANDJAMESM.ENGLANDTheWistarInstituteofAnatomyandBiology,Philadelphia,Pennsylvania19104Receivedforpublication22November1976Rabiesvirus-specificpolypeptidesynthesiswasexaminedunderhypertonicconditions,whichselectivelyinhibitcellularproteinsynthesis.Therabiesvirusproteins(L,G,N,M,,M2)weresynthesizedthroughoutthecourseofinfection,withlittlechangeintheirrelativeratesofsynthesis.TheratesofsynthesisoftheGandM,polypeptidesweremoresensitivetoincreasingosmolaritythanthoseoftheL,N,andM2polypeptides.Extrapolationtoisotonicityoftheresultsobtainedunderhypertonicconditionsindicatedthatthemolarratiosofthepolypeptidessynthesizedundernormalconditionswere0.4(L),64(G),100(N),75(M,),and35(M2).Ahigh-molecular-weightpolypeptide(-190,000),desig-natedpolypeptideL,wasrepeatedlydetectedbothininfectedcellsandinextracellularvirus.TheestimatednumberofLpolypeptidemoleculespervirionDownloadedfromwas33.Thesynthesisofaviralglycoproteinprecursor,designatedgp78,pre-cededtheappearanceofthematureviralglycoproteinininfectedcellslabeledwith[3H]glucosamineunderisotonicconditions.Incellslabeledunderhyper-tonicconditions,littleornomatureviralglycoproteinwasdetected,butavirus-specificglycoproteinwithanelectrophoreticmobilitysimilartothatofgp78wasobserved.Thisglycoproteincouldbechasedintomatureviralglycoproteinwhenjvi.asm.orgthehypertonicconditionsweremadeisotonic.Theseresultssuggestthatareversibleblockofviralglycoproteinsynthesisoccursunderhypertoniccondi-tions.byonJanuary29,2010Rabiesvirus,arhabdovirus,isanegative-TheRNAofthisnegative-strandviruswouldstrandvirusthatcontainssingle-strandedRNAappeartorequireavirion-associatedRNAtran-withamolecularweightofapproximately4.6xscriptasefortranscription(2,21).TheLpro-106(40,43).TheRNAisassociatedintheviriontein,whichispresentinsmallamountsinin-withphosphorylatednucleoprotein(N),form-tactVSVvirions(16,50,51),hasbeenshowntoinganucleocapsidcore(40,41).Aviralenve-benecessaryforRNAtranscriptaseactivity(5-lope,whichcontainstwonon-glycosylated7,10,11,31).Incontrast,thepresenceofLmembraneproteins(M,andM2)andaglyco-proteininrabiesvirionshasnotbeenclearlyprotein(G),surroundsthenucleocapsid(40).established(40,42),andRNAtranscriptaseac-Theviralglycoproteinisresponsibleforelicit-tivityhasnotbeendetectedinpurifiedrabiesingtheproductionofneutralizingantibodyvirions(41,48),exceptpossiblyatextremelyagainstthevirus(53).lowlevels(D.Bishop,personalcommunica-Rabiesvirushasaneclipseperiodof6to12htion).Althoughvirus-specificRNAtranscrip-inBHK-21cellsandproducesvirusforseveraltaseactivityhasbeendetectedinrabies-in-dayswithoutappreciableinhibitionofcellularfectedcells(48;H.P.MadoreandF.Sokol,DNA,RNA,orproteinsynthesis(12,27,47,unpublishedobservations),thevirus-specific52).Evidenceofviralpolypeptidesynthesisinpolypeptide(L),whichmaybenecessaryforrabiesvirus-infectedcellshasbeenlimitedtoviralRNAtranscriptaseactivity,hadnotbeenthedetectionofvirus-specificpolypeptidesindetectedinourpreviousanalysisofinfectedthecytoplasmofinfectedcellsbyimmunoflu-cellslabeledunderhypertonicconditions(26).orescenttechniques(47,52)andbytheisolationInthisreport,hypertoniclabelingconditionsofnucleocapsidcoresaftercellfractionation(8,wereemployedtocharacterizerabies-specific39).Recently,wereportedthatthesynthesisofpolypeptidesynthesisduringthecourseofthemajorrabiesstructuralproteins(G,N,M1,BHK-21cellinfection.InadditiontothemajorM2)couldbedetectedininfectedcellslabeledviralstructuralpolypeptides,ahigh-molecular-withradioactiveaminoacidsunderhypertonicweightpolypeptide(L)hasbeenclearlyidenti-conditions(26).fiedbothininfectedcellsandinextracellular102 VOL.22,1977RABIESPROTEINSYNTHESIS103virus.TheeffectofincreasedosmolarityonMEM(290mOsM),followedbyachasewith2mlofratesofsynthesisofindividualviralpolypep-0.2%BSA-MEM(290mOsM)for24hat370C.tidesandonglycosylationofviralglycoproteinPreparationofcellularfractions.Cellslabeledwasalsoexamined.underhypertonicorisotonicconditionswerewashedtwicewithice-coldNTbuffer(0.13MNaCl,0.05MMATERIALSANDMETHODSTris-hydrochloride,pH7.8).Whole-celllysateswereCellsandvirus.BHK-21/C13cells(25)wereseri-preparedbyscrapingthecellsfromthetissuecul-allypassagedinBlakebottlesandmaintainedinturedishwith2mlofice-coldNTbufferandsonicat-minimalessentialEaglemedium(MEM),supple-ing(Accousticon)for1min.Nuclearandcytoplas-mentedwith10%fetalcalfserum(growthmedium)micfractionswerepreparedafterDouncehomogeni-at370C(18).Rabiesvirus(ERAstrain)andvesicularzation,aspreviouslydescribed(26).Totalproteinstomatitisvirus(VSV)(Indianastrain)wereob-contentwasdeterminedbyamodifiedLowryproce-tainedfromTadeuszWiktoroftheWistarInstitute.dure(30),andtrichloroaceticacid-precipitablera-StocksofrabiesvirusandVSV(108to109PFU/ml)dioactivitywasdeterminedbyprecipitationofsam-werepropagatedinBHK-21cellsinamannersimi-plesinice-cold10%trichloroaceticacid,followedbylartothatdescribedforthepreparationoflabeledwashingwithice-cold5%trichloroaceticacidonvirus.Bothcellsandvirusstockwereshowntobeglass-fiberfilters(WhatmanGF/C).mycoplasma-freebytheassayofSedwickandWik-Preparationoflabeledvirus.Rollerbottlesofcon-tor(38).fluentBHK-21cells(1.5x108cells/bottle)wereInfectionandmockinfectionofcells.Thegrowthinfectedwith5PFUofrabiesvirus(ERAstrain)permediumwasremovedfromconfluentmonolayersofcellor0.01PFUofVSV(Indianastrain)percell.BHK-21cellsin60-or100-mmtissueculturedishes,Viruswasadsorbedfor30minat330Cin10mlofDownloadedfromandthecellswereinfectedwith50to100PFUofinoculum,andthen100mlof0.2%BSA-MEMwasrabiesvirus(ERAstrain)percellin1mlormock-addedtoeachbottleforincubationat330C.A14C0infectedwithanequalvolumeofMEMsupple-labeledaminoacidmixture(0.5,uCi/ml,NEC-445;mentedwith0.2%bovineserumalbumin(0.2%15aminoacids,NEN)wasaddedtotherabies-in-BSA-MEM).Viruswasadsorbedfor30minat370Cfectedcells,or[14C]leucine(0.5ACi/ml,NEC-279,beforeunadsorbedvirusandmockinfectionmedium270mCi/mmol,NEN)wasaddedtotheVSV-in-wereremoved.Bothcellculturetypeswereincu-fectedcells.Therabies-infectedcellswereharvestedjvi.asm.orgbatedingrowthmediumat370Cina5%C02atmos-after4to5daysofincubationat330C;theVSV-phere.infectedcellswereharvestedafter12to16hofLabelingofvirus-infectedandmock-infectedincubationatthesametemperature.Theculturecells.Growthmediumwasremoved,andeachcul-mediumcontaininglabeledviruswasclarifiedbybyonJanuary29,2010turewasincubatedinEarlesaltsolutionplus10%centrifugationat450xg.,for15minat40C,andthefetalcalfserumat3700for15mintodepleteaminoviruswaspurifiedbyamodificationoftheprocedureacidpools.CellslabeledunderisotonicconditionsdescribedbyB.Dietzschold,J.H.Cox,andL.G.wereincubatedforvarioustimesat370CinEarleSchneider(manuscriptinpreparation).Inbrief,thesaltsolutionplus10%fetalcalfserumwith40,uCiofviruswaspelletedfromtheclarifiedsupernatantby3H-labeledaminoacidmixtureperml(NET-250,15centrifugationinaBeckmantype19rotorat19,000aminoacids,NewEnglandNuclearCorp.[NEN])orrpmfor120minat40C.Theviruspelletwasresus-10,ACiof[3H]leucineperml(TRK170,50Ci/mmol,pendedwithaPasteurpipetteinNTE(0.13MNaCl,Amersham/Searle).Cellslabeledunderhypertonic0.05MTris,pH7.8,0.001MEDTA)bufferandconditionswereincubatedfor15minat370Cinlayeredona10to60%sucrosegradientinNTEvarioushypertonicmediacontainingdifferentbuffer.ThegradientwascentrifugedinaBeckmanamountsofexcessNaCl(toallowrunoffofcellularSW27rotorat22,000rpmfor90minat40C.Thevi-mRNAfrompolysomes[35])inEarlesaltsolutionrusbandwascollected,dilutedinNTEbuffer,andplus10%fetalcalfserumandthenlabeledforvar-repelletedbycentrifugationinanSW27rotoratioustimeswithoneoftheaboveisotopesat370Cin20,500rpmfor90minat40C.Theviruspelletwashypertonicmedia.IdenticalresultswereobtainedresuspendedinasmallamountofNTEbufferover-whenlabelingwasdoneunderhypertonicconditionsnightat40C,andthenclarifiedbycentrifugationatwith[3H]leucineor3H-aminoacidmixture(after800xg.,for10minat40C.Labeledvirusreleasedcorrectingfordifferencesintherelativeleucinecon-intotheculturemediumduringthe24-hchase(Fig.tentoftheindividualviralpolypeptides).Cellswere6c)waspurifiedbylayeringthesupernatant(2ml)labeledwith50,uCiof[3H]glucosaminepermldirectlyontoa10to50%sucrosegradientinNTE(NET-190,12Ci/mmol,NEN)inisotonicorhyper-bufferandcentrifugingasdescribedabove.tonic0.2%BSA-MEMaccordingtotheprocedurePolyacrylamidegelelectrophoresis(PAGE).Cellpreviouslydescribedforaminoacidlabeling.ThelysatesandvirussampleswerepreparedforPAGEtonicity(milliosmolarity)ofthevariousmediawasaftertrichloroaceticacidprecipitation,asdescribeddeterminedwithanautomaticosmometer(Osmette-bySokoletal.(44),orprecipitationin5volumesofA,PrecisionSystems,Inc.,Sudbury,Mass.).absoluteethanol.Thesamples(in8Murea)wereInpulse-chaseexperiments,cellsin60-mmtissuesubjectedtoelectrophoresisincontinuous7.5%poly-culturedisheswerepulse-labeledwith100,Ciofacrylamidegelsin0.1Mphosphatebuffer(pH7.2)[3H]leucineinhypertonicmedium(550mOsM)for2containing0.1%sodiumdodecylsulfate(SDS)(44),handthenwashedtwicewith5mlof0.2%BSA-orondiscontinuous10%SDS-polyacrylamidegels 104MADOREANDENGLANDJ.VIROL.(23).Thegelswerefixedin25%isopropanol-10%aceticacid,cutinto1-mmslices,andthenincubated100-overnightat330Cin5mlofa5%Protosol-liquidscintillationfluidmixture.080RESULTSRelativerateofviralpolypeptidesynthesisawininfectedBHK-21cellsunderhypertonic460-conditions(550mOsM).Theincorporationof0.0z3H-labeledaminoacidsintoviralpolypeptideswa.)0wasmeasuredunderhypertonicconditions(550I.mOsM)ininfectedcellsduring1-hlabeling0.0.40Qperiodsat6,12,24,36,48,and72hafterzEinfection.Analysisofthecytoplasmicandthea)nuclearfractionsonPAGErevealedthatabout20-85%ofthelabeledrabies-specificproteinswereinthecytoplasmicfraction,aspreviouslyde-scribed(26).Fromthesegels,therelativerateof3H-labeledaminoacidincorporationintotheI020304050607080viralpolypeptidespermilligramoftotalcellu-TIMEAFTERINFECTION(h)Downloadedfromlar(nuclear+cytoplasmic)proteinperhourFIG.1.Rateof3H-labeledaminoacidincorpora-wascalculated(Fig.1).Duringthe1-hlabelingtionintorabiesviruspolypeptidesunderhypertonicperiod,3H-labeledaminoacidincorporationconditions(550mOsM).Confluentmonolayersofintoindividualviralpolypeptideswaslinear.BHK-21cells(5X106cellsperdish)infectedwith50Only1.5to3.0%ofthelabeledpolypeptidesPFUofrabiesviruspercellwerelabeledfor1hatwerefoundinthemedium.Analysisofthese37°Cwitha3H-labeledaminoacidmixture(40puCi/jvi.asm.orgreleasedpolypeptidesbyPAGErevealedthatml)in550mOsMmediumat6,12,24,36,48,andlessthan10%ofthesepolypeptidesco-migrated72hafterinfection.Thecellswerefractionatedintowithviralproteinsandthattherewasnopref-nuclearandcytoplasmicfractions,and400pgoferentialreleaseofanyviralpolypeptideintoproteinfromeachfractionwasrunoncontinuousbyonJanuary29,2010themedium.7.5%SDS-polyacrylamidegels.Radioactivityincor-Inaddition,themediumusedforporatedintotheviralpolypeptidesineachcellularcellslabeledfor2hwith[3H]leucineat550fractionwasobtainedbysubtractingradioactivitymOsMdidnotcontaindetectablelabeledvirusduetoresidualhostpolypeptidesynthesisfromtheparticles(detectionlimit,5%of290mOsMcon-peaksrepresentingtheviralpolypeptides.Finally,trol),asdeterminedbysucrosegradientanaly-thesumofradioactivityincorporatedintotheviralsis(10to50%sucrose).polypeptidesinthenuclearpluscytoplasmicfractions3H-labeledaminoacidincorporationintoG,wasdividedbythetotalproteininthenuclearplusN,andMlpolypeptideswasdetectedby12hcytoplasmicfractionstogivethecountsperminuteafterinfection,andincorporationintoM2poly-incorporatedpermilligramoftotalcellularprotein.Co-electrophoresiswith"4C-aminoacid-labeledra-peptidewasnotedby24hafterinfection/(Fig.biesvirusproteinsidentifiedtheviral-specificpeaks.1).Therateof3H-labeledaminoacidincorpora-tionintothemajorviralpolypeptides(G,N,tide,especially,issynthesizedatarateapprox-M1,M2)increasedgraduallyforatleast72himately170-foldlessthanthatoftheNpolypep-afterinfection.Ifaminoacidsforviralpolypep-tide.Thepresenceofthispolypeptideinin-tidesynthesisareavailablefromasingleaminofectedcellsisattimesobscuredbyotherhigh-acidpoolandtheindividualpolypeptideshavemolecular-weightpolypeptidesatthetopofcomparablestabilities,therelativerateofSDS-polyacrylamidegels.aminoacidincorporationintothesepolypep-Effectofincreasingtonicityonviralpoly-tidesismostlikelyameasureoftheirrelativepeptidesynthesis.TheratesofNandM2poly-rateofsynthesis.InFig.2,therelativerateofpeptidesynthesiswereenhancedrelativetotheaminoacidincorporationintoviralpolypep-ratiosofL,G,andM,polypeptidesynthesistidesunderhypertonicconditions(550mOsM)withincreasingtonicity(Fig.3).Theincorpora-obtainedfromseveralexperimentswasusedtotionofradioactiveleucineintoallviralpolypep-determinetherelativenumberofviralproteintideswasinhibitedastonicityincreased(e.g.,moleculessynthesizedperhourat550mOsM.incorporationintoviralpolypeptidesat550Thesedatashowthatindividualviralpolypep-mOsMis25%ofincorporationat290mOsMtidesarenotsynthesizedinequimolaramounts[26]).Therefore,theenhancementinrateofsyn-underhypertonicconditions.TheLpolypep-thesisofNandM2(ascomparedwiththeother VOL.VOL.22,22,19771977~~~~~RABIESPROTEINSYNTHESIS105numberofpolypeptidemoleculessynthesizedwasnotcomparabletotherelativenumberofU.-i100-polypeptidemoleculespresentinextracellularAAevirus(Table1).05Virus-specifichigh-molecular-weightpoiy-~atpeptide(L)ininfectedcellsandinpurified'a-ivirus.Synthesisofahigh-molecular-weightz25polypeptidewasrepeatedlydetectedbyPAGE0~~~~~~~~~~~~~ininfected(Fig.4a)butnotinuninfectedcells(Fig.4b).Thispolypeptide,designatedtheL102I03I04050607080polypeptide,wasalsopresentinpurifiedrabiesTIMEAFTERINFECTION(h)virions(Fig.5a)andco-migratedwithLpoly-FIG.2.Relativenumberofviralpolypeptidemole-peptideofVSV.Analysisofpurifiedrabiesyiin-culessynthesizedunderhypertonicconditions(550onslabeledwith[3Hlglucosamineand14C-la-m~sM)atdifferenttimesafterinfection.Radioactiv-beledaminoacidsindicatedthattheLpolypep-ity(31-labeledaminoacidor[3H]leucine)incorpo-tideofrabiesviruswasnotglycosylatedandratedintoindividualviralpolypeptidesperhourwasdeterminedinseveralexperimentsby7.5%SDS-polyacrylamidegelanalysisofcellextractsasde-coTscribedinMethods.Correctionsweremadefordiffer-Z50-Downloadedfromencesintherelativeleucinecontentofsteady-state-labeledviralpolypeptides(theratioof3H-labeled0INC.aminoacidmixturef[3H]leucineis:L,0.78;G,1.03;C.)N,1.21;Ml,0.80;M,,0.72).Thepercentageofradio--.40-activityincorporatedintoindividualviralpolypep-tidepeaks(countsperminuteincorporatedintoindi-a.vidualviralpolypeptidepeaks/totalcountspermin-Cl)uteincorporatedintoviralpolypeptidesx100)wasCCjvi.asm.orgconvertedtomoleculesofproteinfromtheirrespectivemolecularweightsandnormalizedto100moleculesofNpolypeptide.Themolecularweightsusedforthe-Jcalculationwere:L,190,000(seetext);G,80,000;N,byonJanuary29,201062,000;M,,40,000;M2,25,000(40).Exceptforo20-the12-handthe36-htimepoints,thevaluesobtainedare--Gderivedfromtwotofourseparateexperiments.0I-0zviralpolypeptides)reflectsarelative,ratherW.)IAthananabsolute,increaseintherateofsynthe-sisoftheseproteins.r-----A-6N1a.Overtherangeoftonicitiesexamined(450toCT----I-II250350450550650650mOsM),theplotsoftherelativerateofsyn-thesismOSMversustonicityoftheindividualviralpolypeptideswerelinear.IfweassumethatFIG.3.Effectofincreasingtonicityonrelativethislinearrelationshipextendstoisotoniccon-ratesofrabiesviruspolypeptidesynthesis.Confluentditions,monolayers(5X106cells/60-mmdish)ofrabies-ashasbeenshownforVSVpolypeptidesynthesisinfected(50PFU/cell)andmock-infectedBHK-21(32),thedatainFig.3,whenextrapo-cellswerelabeledat24hafterinfectionfor1hwithlatedtoisotonicity,giveanestimateoftherela-[3H]leucine(10Ci/ml)underisotonic(290mOsM)tiveratesofsynthesisofindividualrabiesvirusorhypertonic(450,500,550,600,and650mOsM)polypeptidesunderisotonicconditions(Fig.3,conditions.Whole-celllysates(400Mgofprotein)ofdottedlines).Thepercentageoftotalvirus-infectedandmock-infectedcellswererunwith'4C-specificradioactivityincorporatedintoindi-aminoacid-labeledproteinmarkersoncontinuousvidualviralpolypeptidesatisotonicity(2907.5%SDS-polyacrylamidegels.The[3H]leucinein-mOsM)wasestimatedtobe:L=0.5%,N=corporationintotheviralpolypeptidepeaksattnibut-34%,Gabletoresidualcellularproteinsynthesiswassub-=33%,Ml=25%,andM,=8%.Thesevaluestractedafternormalizingthepolypeptidepatternofwereusedtocalculatetherelativenum-berthemock-infectedtotherabies-infectedcells.Thenetofviralproteinmoleculessynthesizedun-[3H]leucineincorporationintovirus-specificpolypep-derisotonicconditions(Table1)andshowthat,tides(aftercorrectionfordifferencesinrelativeleu-underisotonicconditions,theL,G,N,M1,andcinecontentoftheviralpolypeptides)wasconvertedM,polypeptideswerenotsynthesizedinequi-tothepercentageoftotalvirus-specificcountsinthemolaramounts.Furthermore,therelativeindividualviralpolypeptides. 106MADOREANDENGLANDJ.VIROL.TABLE1.Relativenumberofviralpolypeptidethusdidnotappeartobeadimeroftheviralmoleculessynthesizedglycoprotein(Fig.5b).NumberofLproteinmoleculespervirion.ConditionsLGNMIM2AnestimateofthenumberofLproteinmole-Hypertonic(550mOsM)a0.60321003179culespervirionwasmadefromdataderivedIsotonic(290mOsM)b0.40641007535fromPAGEanalysisofthreedifferentprepara-Relativeno.ofviralpoly-1.91011005286tionsofpurifiedrabiesvirusuniformlylabeledpeptidemoleculesperwith"4C-labeledaminoacids(Table2).Approx-virioncimately33LproteinmoleculeswereestimatedaPercentageof3H-labeledaminoacidincorporatedintotobepresentperrabiesvirion.Thenumberofvirus-specificpolypeptidesat550mOsM(Fig.3)convertedeachoftheothermajorstructuralproteinmole-torelativenumberofviralpolypeptidemoleculessynthe-culespervirionestimatedfromthesedatacor-sizedperhour,asdescribedinFig.2.ThenumberofNpolypeptideswassetequalto100.respondedtothevaluesreportedbySokoletal.bExtrapolatedpercentageof3H-labeledaminoacidin-(40).corporatedintovirus-specificpolypeptidesat290mOsMAnalysisofviralglycoproteinbiosynthesis.(Fig.3),convertedtorelativenumberofviralpolypeptideAvirus-specifiedglycopolypeptidelabeledfor2moleculessynthesizedperhour.ThenumberofNpolypep-tidemoleculeswassetequalto100.hwith[3H]leucineinrabies-infectedcellsundercDerivedfromgelanalysesofthreeseparateprepara-hypertonicconditionsconsistentlymigratedintionsofpurifiedrabiesvirus(ERAstrain),uniformlyla-PAGEfasterthanthemarkervirionglycopro-beledwith14C-aminoacids(seetext).ThevalueforNDownloadedfromtein(Fig.6a).Sincethiscomponentwasnotpolypeptidewassetequalto100.detectedinuninfectedcellslabeledundersimi-jvi.asm.orgbyonJanuary29,2010502570co0-0~xI5025405060708090100,FRACTIONNUMBERSTARTFIG.4.Virus-specifichigh-molecular-weightpolypeptide(L)ininfectedcells.(a)Rabies-infected(50PFU/cell)or(b)mock-infectedBHK-21cellmonolayerswerelabeledfor1hwith[3H]leucine(10uCi/ml)underhypertonicconditions(550mOsM)24hafterinfectionat379C,and400-pgportionsofproteinofthewhole-celllysateswereanalyzedondiscontinuous10%SDS-polyacrylamidegels.['4C]leucine-labeledVSVwassubjectedtoco-electrophoresiswiththecelllysates.ThearrowsrefertotheVSVmarkerproteins. VOL.22,1977RABIESPROTEINSYNTHESIS107I050X2IM123QJA2JI040a.)Uzi~%~~4I~~~~~~J31SDownloadedfrom24I-_10203040506070809010011o+jvi.asm.orgSTiRTFRACTIONNUMBERFIG.5.Virus-specifichigh-molecular-weightpolypeptide(L)inpurifiedrabiesvirus.RollerbottlesofBHK-21cells(1.5x108cellslbottle)wereinfectedwithrabiesvirusat5PFU/cell;theinfectedcellswerebyonJanuary29,2010grownin0.2%BSA-MEMfor4daysat33°Cinthepresenceof[3H]leucine(2pmCi/ml)alone,or[3H]glucosamine(10pXilml)and["4C]leucine(2.5pCi/ml).TheviruswasharvestedandpurifiedasdescribedinMaterialsandMethods.(a)Analysisof[3H]leucine-labeledrabiesviruswith[14C]leucine-labeledVSV(12.5-cmgel).(b)Analysisof[3H]glucosamine-and[14C]leucine-labeledrabiesvirus(11-cmgel).TABLE2.Numberofpolypeptidemoleculesperlarconditions(notshown)andcouldbequanti-rabiesvirionatativelyprecipitatedwithmonospecificantise-Poly-~~~~~~~DaltonsMole-rumdirectedagainstpurifiedvirionglycopro-peptid%Totalradio-Ml(oyp-culestein(B.Dietzschold,H.P.Madore,andJ.M.speciesactivitytide/virion)ion)i/virEngland,unpublishedobservations),itwasconsideredtobevirusspecific.Whenrabies-L1.85+0.94190,0006x10633infectedcellsG42.67+1.3080,000145x1061,815werelabeledfor2hwithN32.94±0.4362,000112x1061,806[3H]leucineunderhypertonicconditionsandM,11.17±0.1940,00038x106951chasedfor24hinisotonicmedium,alargeM211.36±0.7325,00039x1061,547proportionoftheradioactivitythatwasprevi-aPercentageoftotalvirion-associated14C-labeledaminoouslyassociatedwiththefaster-migratingin-acidincorporatedintotheindividualviralpolypeptideswastracellularglycopolypeptideshiftedtothedetermined.Thenumberofdaltonsofeachviralpolypep-slowermigrationrateofthevirionglycoproteintidepervirionwasdeterminedbymultiplyingthepercent-(Fig.6b).Asmallproportionoftheradioactiv-ageofeachviralpolypeptideintheviriontimesthemolecu-larweightofthetotalpolypeptide(340.4x106daltons)inityassociatedwiththeintracellularglycopoly-thevirion(calculatedfromtheratioofpolypeptidetoRNApeptidestillmigratedmorerapidlythanthe[74:1]inthevirion,assumingtheRNAmolecularweighttovirionglycoprotein(Fig.6b).The[3H]leucine-be4.6x106daltons[40]).Thenumberofeachpolypeptidelabeledglycopolypeptidemoleculepervirionwascalculatedbydividingthenumberthatwassynthesizedofdaltonsofeachpolypeptidepervirionbythemolecularunderhypertonicconditionswasincorporatedweightofthatpolypeptide.Themolecular-weightvaluesforintovirusreleasedintothemediumduringthetheG,N,M,,andM2arefromSokoletal.(40).The24-hchaseinisotonicmedium,asshownbyco-molecularweightoftheLproteinwasestimatedat190,000migrationwiththerabies14C-labeledonthebasisofco-electrophoresiswiththeVSVLproteinvirion(Fig.5a)(49).glycoproteinthatwaslabeledinisotonicme- 108MADOREANDENGLANDJ.VIROL.dium(Fig.6c).Theseresultsindicatethathy-pertonicconditionscausetheaccumulationofamorerapidlymigratingformoftheviralglyco-protein.Duringanisotonicchase,however,thismorerapidlymigratingformofglycopro-teinisconvertedtoaformthatco-migrateswithvirionglycoproteinandbecomesassoci-atedwithextracellularvirus.Ineffortstodetectthesynthesisofafaster-migratingformofviralglycoproteinunderiso-tonicconditions(290mOsM),infectedandunin-fectedcellswerelabeledwith[3H]glucosamine.AsshowninFig.7aandb,aprominenthetero-geneouspeakof[3H]glucosaminelabelwasde-tectedintheinfectedbutnotintheuninfectedcelllysates.Thisviral-specificglycopolypeptideshowedtwocomponentsonthegel,amajorcomponent,whichco-migratedwiththevirionN0201A000glycoprotein,andaminorcomponent,whichmigratedmorerapidlyasashoulder.Bothcom-Downloadedfromc.Ic-)ponentscouldbequantitativelyprecipitated25M-Lwithantibodydirectedagainstvirionglycopro-Wu10-IS50tein(B.Dietzschold,H.P.Madore,andJ.M.Z~~~~~~~~0England,unpublishedobservations).Wehavedesignatedthemorerapidlymigratingcompo-nentgp78todistinguishitfromtheglycopro--------r---jvi.asm.orgFteinpresentinextracellularvirus(gp8O).Thefaster-migratingcomponentexhibitedapproxi-35-matelythesameelectrophoreticmobilityasthevirus-specificpeaklabeledwith[3H]glucosa-byonJanuary29,201030-mineunderhypertonicconditions(Fig.7candd).25-Toexaminethepossibilitythatgp78wasan1020~~340607intermediateinthebiosynthesisofrabiesvirus206P10u0glycoprotein(gp8O),infectedcellswerepulse-ti5(05labeledwith[3H]glucosamineunderisotonicconditionsforintervalsfrom30minto4h(Fig.i0(0PK8).After30minoflabeling,themajorityof[3H]glucosamine,whichwasassociatedwithvi--2.5Irus-specificglycopolypeptide,wasinthefaster-migratingcomponent,gp78(Fig.8a).After1h10203040506070°oflabeling,aportionofthe[3H]glucosamineSATFRACTIONNUMBERlabelwasinashoulderofthegp78peakthatmigratedwiththegp8Omarker(Fig.8b).AfterFIG.6.Pulse-chaseanalysisofintracellularviral2h(Fig.8c)and4h(Fig.8d)oflabeling,theglycoproteinsynthesizedunderhypertoniccondi-majorityofthe[3H]glucosaminelabelwasasso-tions.Cultures(5X106cells/60-mmdish)ofrabies-ciatedwithgp8O.Thisshiftof[3H]glucosamineinfected(50PFU/cell)BHK-21cellsgrownat370Cfor24hwereeitherpulse-labeledwith[3H]leucinelabelfromgp78togp80withincreasedlabeling(100gCi/ml)for2hat550mOsMat370C(a)ortimesuggeststhatgp78isaprecursorofthepulse-labeledfor2hat550mOsMandthenchasedmatureviralglycoprotein(gp8O).for24hat37°Cin290mOsMmedium(b).Whole-celllysatescontaining50,000trichloroaceticacid-DISCUSSIONprecipitablecpm(a)and(b)andextracellularvirusUsinghypertonicconditionstoselectivelyfrom(b)abovecontaining30,000trichloroaceticacid-precipitablecpm(c)wereanalyzedondiscontin-suppresscellularproteinsynthesis,wehaveuous10%SDS-polyacrylamidegels.Thearrowsin-characterizedseveralaspectsofrabiesvirusdicate14C-aminoacid-labeledrabiesvirusmarkerproteinsynthesisinBHK-21cells.Therabiesproteinssubjectedtoco-electrophoresiswiththesam-virusstructuralpolypeptidesL,G,N,M,,M2ples.aresynthesizedcontinuallythroughoutthe VOL.22,1977RABIESPROTEINSYNTHESIS1091YI200NI.0-w20.00z404ISC-)en0Downloadedfrom-30jvi.asm.org-15102030405060708090160102030405060708090100byonJanuary29,2010FRACTIONNUMBERFIG.7.Labelingofrabies-infectedoruninfectedcellswith[3H]glucosamineunderisotonic(290mOsM)orhypertonic(550mOsM)conditions.Confluentmonolayers(5x106cells/60-mmdish)ofBHK-21cellswereeitherinfectedwithrabiesvirus(100PFU/cell)(aandc)ormock-infected(bandd)andincubatedat37'Cfor40h.Cellswerethenlabeledunderisotonicconditions(290mOsM)for2hwith[3H]glucosamine(50XciIml)(aandb).Afterpretreatmentfor15minwithhypertonicmedium(550mOsM),othercellswerelabeledfor2hinthesamehypertonicmediumcontaining[3H]glucosamine(50puCi/ml)(candd).Thewhole-celllysatescontaining27,000trichloroaceticacid-precipitablecpmwereanalyzedondiscontinuous10%SDS-polyacrylamidegels.Arrowsindicate14C-aminoacid-labeledrabiesvirusproteins.courseofinfection.IncontrasttothesynthesisrateofsynthesisofagivenviralpolypeptideoftheNSpolypeptideofVSV,whichhasbeenandarate-limitingstepinviralassembly.showntobepreferentiallysynthesizedearlyinLodish(24)hasproposedamodelfortheregu-infection(17),noneoftherabies-specificpoly-lationofa-andf3-globinsynthesiswhichpre-peptidesappearedtobesynthesizedatagreaterdictsthatareductionintheoverallrateofrelativerateearlyininfection.peptidechaininitiationwillcausethepreferen-TherelativeratesofsynthesisoftherabiestialtranslationofmRNAspecieswithhigherviruspolypeptidesdonotcorrespondtotheirrateconstantsofpeptideinitiation.Sincehy-relativeproportionsintheintactvirion.Thispertonicconditionshavebeenshowntospecifi-observationindicatesthattheproteincomposi-callysuppresspeptidechaininitiation,thistionofvirionsisnotsolelydeterminedbythemodelcanaccountforthedifferentialinhibi-relativerateatwhichtheviralpolypeptidesaretioncausedbyhypertonicconditionsofthesyn-synthesizedandsuggeststhatadditionalmech-thesisofvariouscellular(33)andviral(rabies,anismsunderlieviralassembly.IntheabsenceVSV[32],andMMTV[36])polypeptides.ofdatathatcomparetheabsoluterateofsyn-Ifthismodelisindeedcorrect,thedifferentthesisofagivenpolypeptidetotherateatsensitivitiesamongthesynthesesofL,N,andwhichthatpolypeptideisincorporatedintovi-M2,aswellasGandMl,polypeptidestohyper-rus,noconclusioncanbemadeconcerningthetonicconditionssuggeststhatatleastthree 110MADOREANDENGLANDJ.VIROL.I30min8.0-1.0-5ft7-5.0IIIITIIII-IIII--2.5N,0oXXa-(0)zbIhf200E