Rapid, High Level Protein Production Using DNA-based Semliki

Vol.273,No.29,IssueofJuly17,pp.18060–18066,1998

©1998byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc.PrintedinU.S.A.

Rapid,HighLevelProteinProductionUsingDNA-basedSemlikiForestVirusVectors*

(Receivedforpublication,January30,1998,andinrevisedform,May1,1998)

DavidP.DiCiommo‡§¶andRodBremner‡ʈ**

Fromthe‡EyeResearchInstituteofCanada,Toronto,OntarioM5T2S8andthe§DepartmentofMedicalGeneticsandMicrobiologyandʈDepartmentsofOphthalmologyandLaboratoryMedicineandPathobiology,UniversityofToronto,Toronto,OntarioM5G1L5,Canada

SemlikiForestvirus(SFV)vectorscanbeproducedcells.Thebaculovirusandvacciniasystemsrequirescreeningfaster,andhaveawiderhostrange,thanbaculovirusofvirusesgeneratedbyhomologousrecombination(2,3),whichvectors.However,theoriginalSFVsystemrequiresincanbetime-consuming,especiallyiflargenumbersofgeneticvitromanipulationofRNA.Wehavegeneratedasystemmutantsarerequired.

thatiswhollyDNA-based.Boththerepliconvector,en-codingSFVpolymeraseandtheproteinofinterest,andToovercometheseobstacles,alphavirusvectorshavethehelpervector,encodingviralstructuralproteins,emergedasusefultoolsinheterologousproteinproduction.TheweremodifiedsothatexpressionwasRNApolymeraseSindbisvirusandSemlikiForestvirus(SFV),1membersoftheII-dependent.TransfectionofthemodifiedrepliconTogaviridaefamily,arethebeststudiedalphaviruses(4).Bothplasmidalonegenerated20–30-foldmoreproteinthanhavebeenharnessedintogeneexpressionsystemsbecauseofobtainedfromasimpleexpressionvector.Expressiontheirself-amplifyinggenomesthatrequireonlythehosttrans-requiredtheSFVreplicase,whichamplifiesrepliconlationalmachinerytoreplicate(5,6).

RNA.TheSFV-basedvectorgenerated10–20-foldmoreInitsnormalreplicationcycle,SFVinfectsthecellandproteinthanaplasmidbasedonSindbisvirus.Cotrans-replicationcommenceswithtranslationofthe5Јtwo-thirdsoffectionofSFVrepliconandhelpervectorsgeneratedthepositive-sensegenomicRNAintoapolyprotein,withsub-viraltitersofaround106infectiousparticles/ml.Asinglesequentautoproteolyticcleavageintofournon-structuralpro-electroporation,platedonone10-cmplate,generatedteins(nsPs),nsP1tonsP4,forminganRNAreplicase(Fig.1A).enoughvirus(107particles)toproduce>500␮gofpro-Thiscomplextargetsareplicationsequenceatthe3Јendofthetein.Wildtype,replicationproficientviruswasnotde-RNAgenome,andreplicatesfull-length(Ϫ)-strandRNAfromtectedinthreetestsutilizingalmost108viralparticles,a(ϩ)-strandgenomicRNAandviceversa.Thestructuralpro-distinctadvantageoveraDNASindbis-basedsysteminteins(sPs)requiredforamaturevirionareencodedbythelastwhichoverhalfthevirusparticlesgeneratedarefullyone-thirdoftheSFVgenome(Fig.1A).Thisregionistran-infectious.ThenewSFVvectorssignificantlyenhancescribedintoasub-genomicmessagebytheSFVreplicase,andtheutilityofthisexpressionsystem.

translatedintoapolyproteinthatisautoproteolyticallycleaved,producingthecapsidproteinandtwoenvelopeglyco-proteins(Fig.1A).ThecapsidproteinrecognizesapackagingAfundamentaltechniqueinbothbasicandappliedmolecu-signalburiedinthecodingregionfornsP2and,togetherwithlarbiologyistheproductionofheterologousproteins.Bacterialthefull-lengthRNA,formsanucleocapsid(7–9).Thisstructuresystemsarewidelyutilized,butarehamperedbytheinabilityinteractswiththeenvelopeproteins’cytoplasmicdomainsandtomodifyeukaryoticproteinsappropriately.Inaddition,eu-budsfromtheplasmamembranetoformamature,infectiouskaryoticcellshaveevolvedspecificmechanisms,whicharevirus.

lackinginbacteria,tofoldmultidomainproteins(1).BacterialTheoriginalSFVexpressionsystememploysaplasmidinhosts,therefore,areconsiderablylessefficientatproducingwhichtheSP6RNApolymerasepromoterliesupstreamofthewellfolded,soluble,biochemicallyactiveproteinthanmamma-cDNAversionoftheSFVgenome,modifiedsuchthatthesPliancells.

codingregionhasbeenreplacedbythegeneofinterest(lacZinGeneexpressioninmammaliancultureisimpededbypoorFig.1,BandC).“Replicon”RNA(re-RNA)istranscribedandtransfectionefficiency,limitedhostcellrange,andthecom-cappedinvitro,andtransfectedintomammaliancellswhereitplexityoftheexpressionsystem.Recombinantvirusesaretheisamplified.Largeamountsoftargetproteinaregeneratedmostefficienttoolsforproteinproductioninhighereukaryoticfromthesubgenomicmessage.Infectiousparticlescarryingcells.Baculovirusvectorscanproducelargeamountsofheter-re-RNAcanbegeneratedinvivobycotransfectionof“helperologousprotein,bututilizeinsecthostcells,whichhavebeenRNA”(5,10).HelperRNAencodestheviralstructuralproteinsshowntoglycosylateproteinsdifferentlythanmammalian

foundinthewildtypeSFV,butlacksthepackagingsignal(⌿)foundinthensP2openreadingframe.Asaresult,onlyre-RNA*ThisworkwassupportedbytheNationalCancerInstituteofCan-ispackagedwithinawildtypeviralcoat,generatingarecom-adawithfundsfromtheCanadianCancerSociety,theVirginiaS.BoyceResearchFundofPreventBlindnessAmerica,andtheRPResearchFoundation-FightingBlindness.Thecostsofpublicationofthisarticle1Theabbreviationsusedare:SFV,SemlikiForestvirus;nsP,non-weredefrayedinpartbythepaymentofpagecharges.Thisarticlemuststructuralprotein;sP,structuralprotein;re-RNA,repliconRNA;RPV,thereforebeherebymarked“advertisement”inaccordancewith18replicationproficientvirus;CMVIE,cytomegalovirusimmediateearly;U.S.C.Section1734solelytoindicatethisfact.

X-gal,5-bromo-4-chloro-3-indolyl-␤-D-galactoside;PAM,plaqueassay¶RecipientofaMedicalResearchCouncilofCanadastudentship.medium;PFU,plaque-formingunit;RSVLTR,Roussarcomaviruslong**Towhomcorrespondenceshouldbeaddressed:EyeResearchIn-terminalrepeat;m.o.i.,multiplicityofinfection;BHK,babyhamsterstituteofCanada,399BathurstSt.,Toronto,OntarioM5T2S8,Canada.kidney;PBS,phosphate-bufferedsaline;FBS,fetalbovineserum;F,Tel.:416-603-5865;Fax:416-603-5126;E-mail:rbremner@playfair.farad(s);kb,kilobasepair(s);IMDM,Iscove’smodifiedDulbecco’sme-utoronto.ca.

dium;ONPG,O-nitrophenyl-␤-D-galactopyranoside.

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binantvirusthatiscapableofoneroundofinfection.

SeveralfeaturesoftheSFVhavemadeitausefulvectorfortheexpressionofforeigngenes.First,andincontrasttobacu-lovirus,SFVwillinfectalmostanymammaliancell.Second,itispossibletogeneratereasonableamountsofproteinsimplybytransfectingre-RNA(10).Thisapproachavoidsgeneratinganyvirus;however,itislimitedbytransfectionefficiencyandre-quireslargeamountsofRNA.Third,viruscanbeproducedquicklyandefficientlyin2days(10),whichmakesitthefastestsystemavailable.Fourth,amplificationbySFVreplicaseissoefficientthattheleveloftargetproteincanreachupto25%oftotalcellularprotein(10).Finally,mutationswithintheglyco-proteingenep62(Fig.1A)preventinfectionunlessthevirusisproteolyticallytreatedwith␣-chymotrypsin(11).Thissafe-guardfeaturesignificantlydecreasesthelikelihoodofgenerat-ingreplicationproficientvirus(RPV)sincetwoeventswouldberequired;recombinationbetweenre-RNAandhelperRNA,andreversionorsuppressionoftheconditionalmutation.SFVvec-torshavebeenusedtogenerateseveralrecombinantproteins(see,forexample,Refs.10,12,and13;reviewedinRef.14),toproducehybridviruses(15,16),andtostudyaspectsoftheSFVlifecycle.

TheoriginalSFVexpressionsystem(Fig.1B)ishamperedbythenecessitytogeneratecappedRNAtranscriptsinvitroandrequiresspecializedconditionsforRNAhandling.Toovercometheseobstacles,weconstructedaDNA-basedself-amplifyingSFVvectorbyreplacingtheSP6promoterusedintheoriginalsystemwiththeRNApolymeraseII-dependentcytomegalovi-rusimmediateearly(CMVIE)enhancer/promoter,whichdrivestranscriptioninvivo(Fig.1B).Transfectionofthisvec-torintoBHKcellsgeneratedhighlevelsofprotein(20–30pg/cell),productionofwhichwasdependentonafunctionalSFVpolymerase.TocompletetheDNA-basedexpressionsys-tem,ahelperplasmidwasconstructedthatencodesSFVsPs.Cotransfectionofrepliconandhelperplasmidsgeneratedcon-ditionallyinfectiousviruscapableofproducingproteinatthesamehighyieldasvirusgeneratedbytheRNAapproach.NoRPVwasdetectedinseveralrecombinantSFVvirusprepara-tions,adistinctadvantageoveraDNA-basedSindbissystemreportedpreviously(17).

EXPERIMENTALPROCEDURES

Plasmids—pSCA␤wasbuiltinthreestages.(i)TheSV40poly-adenylationsignalwasamplifiedbypolymerasechainreaction(PCR)usingSVhRB(18)asatemplate,VentDNApolymerase,andthefol-lowingprimers:O-SV1(SpeI):5Ј-gccgactagtgatcataatcagccata-3ЈandO-SV2(XbaI):5Ј-gccgtctagatccagacatgata-3Ј.The0.25-kbproductwascutwithSpeIandXbaI,andligatedintoSpeI-cutpSFV1(10).Theresultingplasmid,pSFVpoly(A),containsapoly(A)signaldownstreamofthemultiplecloningsite.(ii)TheSP6promoterandthefirst513basepairsofthe5ЈendoftheSFVgenomewereremovedfrompSFVpoly(A)bydigestionwithSphIandBsiWI(Fig.1C).The10.3-kbfragmentwasligatedwitha0.7-kbSphI-EcoRICMV-T7fragment(generatedbyamplificationanddigestionofbasepairs1570–2186ofpcDNA/NEO(Invitrogen)withtheprimersO-CMV1(SphI):5Ј-ccgccggcatgcgtaat-caattacggggtc-3ЈandO-CMV2(EcoRI):5Ј-gccggaattcaagcttccggtctcccta-3Ј),anda0.57-kbEcoRI-BsiWIfragmentcontainingthefirstpartoftheSFVgenome(generatedbyamplificationanddigestionofbasepairs1–517inpSFV1withtheprimersO-SFV3(EcoRI):5Ј-gccggaattcatggcg-gatgtgtgacat-3Јandreverseprimer,O-SFV2:5Ј-gtacagcgatgttggtgc-3Ј).Theresultingplasmid,pSCA1,containstheCMVIE/T7promotersupstreamoftheSFVrepliconandpoly(A)region.(iii)pSCA␤wasbuiltbyligatinga4.6-kbBglII-SpeIfragmentfrompSFV3-lacZ(5,10)toa9.9-kbBglII-SpeIpSCA1fragment.ThisplacesthelacZgeneimmedi-atelydownstreamofthensP4codingregion,underthecontroloftheSFVsubgenomicpromoter(Fig.1C).⌬pSCA␤wasbuiltbydeletinga2.7-kbSacIIfragmentfrompSCA␤.pSCAHelper(Fig.6A),wascon-structedbyinsertingthe3.6-kbSpeI/AccIfragmentfrompSCA1intothe5.1-kbAccI/SpeIfragmentfrompSFVHelper2(5,11).

CellCultureMaterials—BHK-21babyhamsterkidneycellsandCOS-1cells(SV40transformedGreenmonkeykidney)wereculturedin

Iscove’smodifiedDulbecco’smedium(IMDM)supplementedwith10%fetalbovineserum(FBS),25mMHEPESbuffer,andL-glutamine.Cellswereincubatedinahumidifiedatmosphereof5%CO2inair.

Electroporation—BHK-21andCOS-1cellsweregrownto50%con-fluence,trypsinized,washedoncewithIMDMplusFBS,oncewithPBSandresuspendedat107cells/mlinasterilemediumcomprisingIMDMwith10mMglucoseand0.1mM␤-mercaptoethanol.Atroomtempera-ture,cells(0.8ml)weretransferredtoa0.4-cmcuvette(Bio-Rad),DNAadded,andelectroporationcarriedoutusingtheBio-RadGenePulser.Cellsfromonecuvettewereplatedonfive6-cmplates.Tooptimizethetransfectionefficiency,variationsincapacitance(25–960mF),voltage(0.1–0.4kV),numberofpulses(1or2),anddelaybetweenpulses(0or30s)werecarriedoutin80combinations.Ininitialexperiments,two30-sdelayedpulsesat0.4kVand960mFyieldedthemaximumlacZexpression.However,re-optimizationwasrequired,andinmorerecentexperimentscellsreceivedonepulseat0.4kVand960␮F.Eachdatapointrepresentstheaverageofthreeelectroporations.

␤-GalactosidaseAssay—␤-GalactosidaseactivitywasmeasuredbyanONPG(O-nitrophenyl-␤-D-galactopyranoside,Ampresco)assayasdescribedpreviously(19).␤-Galactosidasestandards(BoehringerMannheim)wereusedtoconvertthereadingsofA420nmfortheONPGassayresultsintonanogramsofprotein.

X-GalAssay—TransfectedBHK-21andCOS-1cellswerestainedbytheX-gal(5-bromo-4-chloro-3-indolyl-␤-D-galactoside,ChemicaAltaLtd.)method(20).

SouthernBlotAnalysis—BHK-21cells(8ϫ106),electroporatedwith2␮gofplasmid,weresplitintofive6-cmdishes(i.e.0.4␮gofDNA/plate).TotalDNAfromoneplatewasisolatedbyphenol-chloroformextraction(19),and15%ofthesamplewasdigestedandrunona0.8%agarosegel,andtransferredtoaGeneScreenPlusmembrane(NENLifeScienceProducts)asdescribedpreviously(21).The3.1-kbBamHIfragmentofpSCA␤,correspondingtothelacZgene,wasrandom-primelabeled(22)with[␣-32P]dCTPandusedasaprobeforhybridization.Hybridizationwasperformedasdescribed(21).Blotswerewashedtwicein2ϫSSCand1%SDS,thentwiceagainin0.1ϫSSCand0.1%SDSat65°C.SignalwasvisualizedandquantitatedusingaMolecularDiagnosticsPhosphorImager.

GenerationofInfectiousSFVParticles—ThetransfectedamountofpSCA␤andpSCAHelperDNAwasoptimizedtogeneratethehighestviraltiter.Induplicateexperiments,pSCA␤(2␮g)andpSCAHelperwereaddedinmolarratiosof1:0.5,1:1,1:2,and1:4,respectively,toelectroporationmediacontaining8ϫ106BHK-21cells.Cellswereelectroporatedandthecontentsofeachcuvetteplatedona10-cmtissuecultureplate.After4h,themediumwasaspirated,cellswashedwithPBS,and8mlofIMDM(plus10%FBS)wasadded.MediumcontainingtheSFVparticleswascollected24and36hafterelectroporationandclarifiedbycentrifugationat2000rpm(SorvallRT6000D)for15minat4°C.Viruswasactivatedwith0.5mg/ml␣-chymotrypsinatroomtemperaturefor45min,andaprotinin(0.38mg/ml)addedtostopproteaseactivity(11).0.2mlofactivatedvirusstockwasaddedto106BHK-21cellson6-cmtissuecultureplates,andincubatedfor45minat37°Cinahumidifiedatmosphereof5%CO2inair.Theinfectiousmediumwasaspirated,cellswashedwithPBS,andIMDM/10%FBSwasadded.ONPGandX-galassayswereperformed18hafterinfection.X-gal-positiveand-negativecellswerecountedin10fieldsviewedunder400ϫmagnification.Totalnumberofcellsonthedishwasobtainedbytrypsinizingaduplicateplateofinfectedcells.ViraltiterwasestimatedbymultiplyingthetotalnumberofcellsbyboththefractionofX-gal-positivecellsandafactortocorrectforthevolumeofvirus-containingsupernatantusedintheassay.

DeterminationofReplicationProficientVirusParticles—Atotalof8ϫ106BHK-21cellsweretransfectedwitha1:1molarratioofpSCA␤(2␮g)andpSCAHelper(1.2␮g)in10separatereactions.At24haftertransfection,0.5mlofthegrowthmediumfromeach10-cmtissuecultureplatewascollectedandviraltiterdetermined(seeabove).Without␣-chymotrypsintreatment,theremaining6mlweredividedamongsixtissuecultureplates(6cm)containing1ϫ106BHKcellsandincubatedfor45min.Thevirus-containingmediumwasaspirated,andthecellswerewashedinPBSandthenoverlaidwithaplaqueassaymedium(PAM)containing0.6%lowmeltagarose(BioShop,Canada)inIMDM(plus2%FBS,10%tryptosephosphate(LifeTechnologies,Inc.),and20mMMgCl2).Thecellswereincubatedfor5daysandcheckedforplaquesdaily.

Cellsinfectedwithactivatedrecombinantvirusweretestedforpro-ductionofRPVs.Atotalof3ϫ107␣-chymotrypsinactivatedinfectiousunits,derivedfromthreeindependentcotransfectionsofpSCA␤andpSCAHelperDNA,wereusedtoinfect2ϫ107BHKcells.After45min

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FIG.1.TheSFVexpressionsystem.A,replicationofwildtypeSFVgenome.Filledboxesrepresentamplificationsequencesateitherendofthegenome.ThepackagingsignalwithinnsP2(␺)isrepresentedbyastippledbox.Thesubgenomicpromoterisshownasanarrow.B,comparisonofRNA-andDNA-basedSFVrepliconvectors.pSFV3-lacZisdescribedinRef.10.Thisschemeshowsthestepsnecessarytoobtainproteinfromtransfectionofre-RNAorplasmidreplicon.Infectiousviruscanbeobtainedbyco-transfectingpSCA␤withpSCAHelper(Fig.6A).C,detailedmapofpSCA␤.Numbersindicatethestartofeachrestrictionsite.

ofinfection,cellswerewashedwithPBS,overlaidwithPAM,andRESULTS

checkedforplaque-formingunits(PFUs)dailyover5days.

TodeterminetheincidenceofRPVingrowthmediaofcellsinfectedReporterGeneExpressioninVitro—Thecurrentexpressionwithactivatedvirus,1ϫ107BHKcellsweredividedamong106-cmsystembasedonSFVrequirestransfectionofRNAthatcon-platesandinfected,intotal,with4.4ϫ106infectiousunitsof␣-chy-tainstheviralreplicasegenesandtheinsertedgeneofinterestmotrypsinactivatedrecombinantvirus.After18h,thegrowthmediumwasclarified,dividedinto10samples,andappliedto10tissueculture(Fig.1B).RNAisobtainedbySP6-driveninvitrotranscriptionplates(6cm)containingatotalof2ϫ107BHKcells.After45minofofanSFVcDNA-geneXcassette.Ourgoalwastosimplifythisincubation,PAMwasoverlaidontocellsandexaminedforPFUsdailysystembycircumventingtheneedforinvitrotranscriptionandfor5days.

RNAhandling.Thus,twomajormodificationsweremadeto

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FIG.2.Productionof␤-galactosidasebypSCA␤.BHK-21cellswereelectroporatedwiththeindicatedamountsofpSCA␤(shownsche-maticallyatthetopleft)and␤-galactosidaseexpressiondetermined48hlater.Time0,inthisandsubsequentgraphs,is4hafterelectro-poration,whencellshadreattachedandthemediumwasreplaced.Errorbarsinthis,andsubsequentgraphs,indicatestandarddeviation.NotethattheamountofDNAperplateis1/5thofthetotalelectropo-rated,becausetransfectedcellsweresplitontofive6-cmdishes.Inthisexperiment,therefore,2␮gofpSCA␤generatedatotalof500ng␤-galactosidase.

theparentvectorpSFV1(5,10).First,theSV40termination/polyadenylationsignalwasaddedimmediatelydownstreamofthe69Aresiduesatthe3ЈendoftheSFVcDNA(23).Themessageexpressedfromthefinalvectorisexpectedtohavetwopoly(A)stretchesseparatedbyashortSV40sequence.Second,theSP6promoterinpSFV1wasreplacedbyahybridCMVIE/T7promoterderivedfromthepcDNAvector(Invitrogen).Thefinalvector,pSCA␤,wascompletedbyinsertionofthereportergene,lacZ,intothemultiplecloningsite(Fig.1).ThisbifunctionalvectorcanbetranscribedinvivoorinvitrousingeithertheCMVIEorT7promoter,respectively.

TodetermineifpSCA␤wasfunctional,theplasmidwastransfectedintoBHK-21cellsand␤-galactosidaseactivitymeasuredincelllysates.Tooptimizetheamountof␤-galacto-sidaseproduced,pSCA␤wastransfectedin5-foldincrements,from0.08␮gupto10␮g.␤-galactosidasewasdetectedineverycase(Fig.2),indicatingthataDNA-basedSFVexpressionsystemisviable.Maximumexpressionwasobtainedfollowingtransfectionof2␮gofDNA(Fig.2),andthisamountwasusedinallsubsequenttransfectionexperiments.Theamountof␤-galactosidaseobtainedperplaterangedfrom100ng(Fig.2)to900nginmorerecentexperiments(datanotshown),depend-ingontransfectionefficiency.ThenumberoftransfectedcellswasdeterminedinseveralexperimentsbyX-galstaining,and,coupledwithdeterminationofenzymeactivity,consistentlyshowedthatpSCA␤generatesaround20–30pgof␤-galacto-sidase/cell,whichissimilartothe80pg/cellobtainedfollowingtransfectionofinvitrogeneratedre-RNA(10).

pSCA␤ExpressesMore␤-GalactosidasethanCMV␤Gal—Thelevelsof␤-galactosidaseobtainedwiththepSCA␤vectorshouldbemuchhigherthanthoseobtainedfromacomparablevectorthatlackstheSFVnsPs.MessagelevelsfromCMV␤Gal(CLONTECH)aredependentonCMVIEpromoteractivityalone.Thus,BHK-21cellsweretransfectedwithpSCA␤andCMV␤Galand␤-galactosidaseactivitymeasuredover5daysaftertransfection(Fig.3A).Theamountof␤-galactosidaseproducedbypSCA␤reachedamaximumonday2,andonday1fortheCMV␤Galvector.Inseveralreplicateexperiments,theexpressionlevelsobtainedwithpSCA␤were,onaverage,20–30-foldgreaterthanCMV␤Gal.SimilardatawereobtainedusingCOS-1cells(datanotshown).

TheelevatedlevelsofexpressionobservedwithpSCA␤ver-susCMV␤Galplasmidismostlikelyduetotheamplificationof

FIG.3.ComparisonofpSCA␤andasimple,CMVIEpromoter-drivenvector.A,BHK-21cellsweretransfectedwith2␮gofpSCA␤(q)or1␮gofCMV␤Gal(E)(anequimolaramount),and1/5thofthecellsplatedona6cmdish.␤-galactosidaselevelsweremeasuredattheindicatedtimepointsafterelectroporation.Thevectorsareshownsche-maticallyatthetopofthegraph.B,SouthernanalysisofDNAisolatedfromBHK-21cells1dayaftertransfectionwithpSCA␤(lanes3–5)orCMV␤Gal(lanes6–8).Analysisofthreeseparatetransfectionsforeachplasmidareshown.pSCA␤andCMV␤GalsamplesweredigestedwithBamHI.Thecontrol(lanes1and2)isBamHI-digestedpSCA␤.BlotswereprobedwiththeBamHIlacZfragmentfrompSCA␤.

themessageencodedbytheSFV-basedvector.However,itwasalsopossiblethatthisplasmid-basedSFVvectortransfectedmoreefficiently.Againstthispossibility,wefoundthatsimilarnumbersofcellswerestainedblueinanX-galassaywhetherpSCA␤orCMV␤Galwastransfected(datanotshown).Asamorequantitativemeasureoftransfectionefficiency,DNAwasextractedfromthesamelysatesthatwereusedtomeasuretheday1␤-galactosidaseactivitiesshowninFig.3A.DNAwasdigested,andtransfectedplasmiddetectedonaSouthernblotbyuseofalacZprobe,andquantifiedonaPhosphorImager.EqualamountsofpSCA␤andCMV␤Galweretransfected(Fig.2B).Therefore,thehigherexpressionobservedwithpSCA␤wasduetotheeffectsoftheSFVpolymerase(Fig.1A).

ReporterGeneExpressionfrompSCA␤IsDependentonFunctionalReplicaseProteins—ToconfirmthattheSFVnsPswereindeedessentialtotheactivityofpSCA␤,wetestedwhetheraderivativewithalargedeletioninnsP2–3coulddirect␤-galactosidaseexpression.Thisplasmid(⌬pSCA␤,Fig.1C)wastransfectedintoBHK-21cellsand␤-galactosidaseactivitymeasuredovera5-dayperiod.pSCA␤expressedanaverageof220ngof␤-galactosidase/6-cmplate,buttherewasno␤-galactosidaseactivityincellstransfectedwith⌬pSCA␤(Fig.4A).Similarly,whenBHK-21cellswerestainedfor␤-ga-lactosidaseusingX-gal,onlypSCA␤-transfectedcellswerepos-itive(Fig.4B).Southernanalysisperformedoncellstakenfrom2,3,and4daysaftertransfectionconfirmedthatbothpSCA␤and⌬pSCA␤plasmidshadbeentakenup(Fig.4C).TheDNAfromeachtransfectionwasquantitatedonaPhosphorImagerandthedatausedtobase-linecorrectthe␤-galactosidaselevels(Fig.4A).Thus,␤-galactosidaseexpressionfrompSCA␤isnotduetoread-throughtranslationofthefull-lengthmessage,butisdependentontheSFVpolymerase,whichcarriesoutbothamplificationandsynthesisofthesubgenomiclacZtranscript(Fig.1A).

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FIG.4.pSCA␤functionrequiresthensPs.A,BHK-21cellsweretransfectedwith2␮gofpSCA␤(q)or⌬pSCA␤(E)(drawnschemati-callyatthetopright)and1/5thofthecellsplatedona6-cmdish.␤after-Galactosidaseelectroporation.activityB,cellswastransfectedmeasuredatwiththepSCAindicated␤or⌬pSCAtime␤pointswerestainedwithX-gal.C,SouthernanalysisofBamHI-digestedpSCA␤(lanes3–5)or⌬pSCA␤(lanes6–8)isolated2–4daysaftertransfectionasindicated.ProcedureswereasdescribedinthelegendtoFig.3andunder“MaterialsandMethods.”ControlDNA(lanes1and2)wasBamHI-digestedpSCA␤.M(lane9)representsasamplefrommock-transfectedcells.

pSCA␤ExpressesMoreProteinthanaDNA-basedSindbisVector—Sindbis,anotheralphavirus,wasengineeredbyWolffandco-workers(24)intoaplasmid-basedvector.Inthissystem,invivotranscriptionoftheSindbisRNAwasachievedbyplac-ingviralcDNAundertranscriptionalcontroloftheRoussar-comaviruslongterminalrepeat(RSVLTR).TheSindbisvec-tor,pSin-nlLacZ,alsoutilizedlacZasareportergene.TocompareexpressionfromtheSFV(pSCA␤)andSindbis(pSin-nlLacZ)DNA-basedvectors,pSCA␤andpSin-nlLacZweretransfectedintoBHK-21cellsand␤-galactosidaseactivitywasmeasuredovera5-dayperiod(Fig.5A).␤-Galactosidaseex-pressionpeakedonday2forbothpSCA␤andpSin-nlLacZ.Inseveralreplicateexperiments,␤-galactosidaseexpressioninBHK-21cellswas10–20-foldhigherforpSCA␤thanforpSin-nlLacZ.Transfectionefficiencyofbothplasmids,asassessedbySouthernanalysis,wassimilar(datanotshown).Thus,higherlevelsof␤-galactosidasewereobtainedusingtheSFVvector.GenerationofInfectiousRecombinantSFVUsingDNA-basedVectors—TogenerateinfectiousvirusparticlesusingDNA-basedSFVvectors,weconstructedpSCAHelper,inwhichex-pressionofhelperRNAisdrivenbytheCMVIEpromoter(Fig.6A).ThisRNAcontainsthesequencesateachendoftheSFVgenomethatmediateamplificationbythensPs,butlacksthepackagingsignallocatedinthensP2gene(Fig.1A).Thus,cotransfectionwiththerepliconvectorwillresultinamplifica-tionofthehelpermessage,transcriptionofthesubgenomic

FIG.5.ComparisonofpSCA␤andtheSindbisvector,pSin-nlLacZ.BHK-21cellsweretransfectedwith2␮gofpSCA␤(q)orpSin-nlLacZ(E)(drawnschematicallyatthetopofthegraph),andone-fifthofthecellsplatedona6-cmdish.␤-Galactosidaselevelsweremeasuredattheindicatedtimepointsafterelectroporation.

message,andtranslationofthesPs.ThesPswillpackagere-RNA,butnothelperRNA.pSCAHelpercarriesmutationsinthep62sPgenethatalterthreeaminoacidssothatgenerationofE2andE3glycoproteinsfromthep62precursorandactiva-tionofthevirusrequires␣-chymotrypsintreatment(11).BHK-21cellsweretransfectedwithpSCA␤andpSCA-Helperplasmidsinfourmolarratios.After24h,0.2mlofthegrowthmediumfromthetransfectedcellswasactivatedwith␣-chy-motrypsin,appliedtofreshBHK-21cells,andtiterdetermined.Thehighestviralyieldwasobtainedwitha1:1molarratio,followedby1:2,1:0.5,and1:4(Fig.6B).

Tooptimizethepostinfectionrecoverytime,BHK-21cellsweretransfectedwithequimolaramountsofpSCA␤andpSCA-Helper.Mediumharvested24hafterelectroporationcontained2.05ϫ106infectiousparticles/ml,whilemediumharvestedat36hafterelectroporationcontained0.80ϫ106infectiouspar-ticles/ml(Fig.6B).Theamountof␤-galactosidasegeneratedafterinfectionwassimilartotheamountsreportedpreviously(10).Fromasingletransfection,platedonone10-cmplate,approximately107infectiousparticlesofviruswasobtained.Infectionof1.1ϫ106BHK-21cellswith3.2ϫ105particlesgenerated16␮gof␤-galactosidase,sothetotalvirusproducedfromthistransfectionwasenoughtogenerateϾ500␮gof␤-galactosidase.

AbsenceofReplicationProficientViruses—Oneoftheprob-lemsassociatedwithanyviralexpressionsystemisthegener-ationofwildtypevirusthroughrecombinationbetweentheseparatedcomponentsoftheviralgenome.Inalphavirussys-tems,replicationproficientvirus(RPV)particleshavebeendetectedfollowingcotransfectionofrepliconandhelperRNAs(11,25).Recombinationoccurswhenthepolymeraseswitchestemplatesduringamplification(26).InaDNA-basedsystem,homologousrecombinationbetweenplasmidsprovidesanotherlevelatwhichacompletevirusgenomecouldbegenerated.However,withtheSFVsystemdescribedhere,recombinationalonewouldnotgeneratefullyinfectiousvirussincereversionorsuppressionoftheconditionalmutationthatinhibitsp62cleavagewouldalsoberequired(11).Weperformedthreeas-saystoestimatethefrequencyofRPVgenerationwiththeDNA-basedSFVsystem(Fig.7).

TodeterminewhetherthegrowthmediafromBHK-21cellstransfectedwithpSCA␤andpSCAHelperDNAcontainedanyRPV,BHK-21cellswerecotransfectedwithpSCA␤(2␮g)andpSCAHelper(1.2␮g)in10separateexperiments.After24h,0.5mlofgrowthmediumwasremovedfromeachtransfectionandusedtodetermineviraltiter.Theremaininggrowthme-dium,whichcontainedover5ϫ107recombinantvirusparti-

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FIG.6.Productionofinfectiousrecombinantvirus.A,diagramofpSCAHelper.Numbersrepresentthestartoftheindicatedrestrictionsites.B,8ϫ106BHK-21cellsweretransfectedwithpSCA␤(2␮g)andpSCAHelperintheindicatedmolaramounts,andallthecellsfromeachtransfectionplatedonone10-cmdish.Viruswasharvested24haftertransfectionandtiterassessedonfreshBHK-21cells18hafterinfec-tion.C,BHK-21cellsweretransfectedwith2␮gofpSCA␤and1.2␮gofpSCAHelper(anequimolaramount),andallthecellsplatedonone10-cmdish.ViralstockswereharvestedattheindicatedtimepointsandtiterassessedonfreshBHK-21cells.

cles,wasaddedto6ϫ107freshBHK-21cellswithout␣-chy-motrypsintreatment.ThecellswereoverlaidwithagaroseandexaminedfortheappearanceofPFUsover5days(Fig.7A).NoPFUsweredetected.

Additionally,noRPVparticleswerefoundwhenplaqueas-sayswereperformedon2ϫ107BHKcellsinfectedwith3ϫ107␣-chymotrypsin-activatedrecombinantviralparticles(Fig.7B).

Furtherexperimentswereperformedtoanalyzeifthecul-turemediumfromcellsinfectedwithactivatedrecombinantviruscontainedanyRPV.BHK-21cellswereinfectedwithactivatedrecombinantvirusovervariousmultiplicitiesofin-fection(m.o.i.).Atotalof4.4ϫ106particleswereusedintheseexperiments.Themediumwascollected18hafterinfection,appliedtonewBHK-21cells,andtestedforthepresenceof

FIG.7.Incidenceofplaque-formingunits(PFUs)duetorepli-cationproficientSFVvirus.A,10individualpSCA␤andpSCA-Helpercotransfectionsgenerated5.3ϫ107infectiousvirusunits,which,without␣-chymotrypsintreatment,wereaddedto6ϫ107freshBHK-21cells.B,threeindividualpSCA␤andpSCAHelpercotransfec-tionsgenerated3ϫ107infectiousvirusunits,whichwereactivatedwith␣-chymotrypsinandaddedto2ϫ107freshBHK-21cells.C,10individualpSCA␤andpSCAHelpercotransfectionsgenerated4.4ϫ106infectiousvirusunits,which,onceactivatedwith␣-chymotrypsin,wereaddedto1ϫ107freshBHK-21cells.After18h,thegrowthmediumfrominfectedcellswasappliedtofreshBHK-21cells.TheinfectedBHK-21cellsinallthreeexperiments(A–C)wereoverlaidwithagaroseandexaminedforPFUsover5days.Theindicatedviraltitersweredeterminedbyinfecting1ϫ106BHK-21cellsfor18hwith␣-chymo-trypsin-activatedvirusobtainedfromtheinitialtransfectionandstain-ingfixedcellswithX-gal.

RPV(Fig.7C).NoPFUsweredetected.

Insummary,atotalof8.7ϫ107virusparticlesobtainedbytransfectionofpSCA␤andpSCAHelperwerenegativeinthreetestsforRPVs,indicatingthatgenerationofwildtypevirusisextremelyrare.

DISCUSSION

Alphavirusexpressionsystemshavebeenusedtoexpressawiderangeofheterologousproteins(reviewedinRefs.14and27).Althoughtheapproachissimplerandmuchfasterthanvectorssuchasbaculovirus,neitherSFVnortherelatedSind-bisvectorshavesupercededbaculovirusastheexpressionsys-temofchoice.Onetechnicaldrawbackoftheoriginalalphavi-russystemsisthattheyrequireproductionandmanipulationofrecombinantRNAinvitro.Therefore,thedesignofDNA-basedalphavirusvectors,bothforSFV(thiswork)andSindbis(17,24),significantlyimprovestheutilityoftheseexpressionsystems.Thesedevelopmentsalsosimplifytheuseofalphavi-rusesasinvivogenedeliverysystems,forexample,inthedeliveryofvaccines(28).

Ourobservations,andthoseofothersusingDNA-basedSindbisvectors(17,24),showthatatleastaportionoffull-lengthre-RNAgeneratedinthenucleusmustreachthecyto-plasm.Indeed,theamountofproteinproducedpercellwiththeDNA-basedSFVvectorwassimilartothatobtainedpreviouslyfollowinginfectionofcellswitharecombinantvirus(10).Ex-pressionwasoptimalwith2␮gofpSCA␤andpeaked48haftertransfection.Withtheseparameters,approximately20–30pgof␤-galactosidasewasobtainedpercell.ProteinexpressionwiththeDNA-basedSFVvectorwas20–30-foldhigherthanwithasimpleCMVIE-basedvector.ThisdifferencecanbeattributedtotheeffectoftheSFVpolymerase,sincealargedeletioninthensPgeneregioncompletelyabolishedproteinexpression.

TwogroupshavedevelopedDNA-basedSindbisexpressionvectors(17,24).WecomparedtheperformanceoftheSindbisvectorpSin-nlLacZ(24)againsttheSFVvector,pSCA␤.pSCA␤Downloaded from www.jbc.org by on April 1, 2007 18066ADNA-basedSemlikiForestVirusExpressionSystem

generated10–20-foldmore␤-galactosidasethantheSindbis-derivedvector.ThisresultisnotduetoadifferenceintheefficiencyofSFVandSindbispolymerases,sincetransfectionofSFVorSindbisre-RNAsyieldssimilarlevelsofprotein(10,27).Thus,othercomponentsofpSCA␤andpSin-nlLacZmustberesponsible.pSin-nlLacZencodes␤-galactosidasethatistar-getedtothenucleus,whereastheproteinencodedbypSCA␤lacksanuclearlocalizationsignal,butitseemsunlikelythatthiswouldexplainthelargedifferenceinexpressionlevels.Animportantelementrequiredformaximalexpressionistheviralpoly(A)tractplaceddownstreamofthereportergene(17).However,bothpSCA␤andpSin-nlLacZcontainsuchamotif.pSCA␤containsanadditionaltermination/polyadenylationsignaldownstreamofthistract,whereaspSin-nlLacZlacksthiselement.However,thismotifinhibitedexpressionofan-otherDNA-basedSindbisvector(24).Otherfactorsthatcouldinfluenceproteinexpressionincludethepromoters(CMVIEorRSVLTRinpSCA␤andpSin-nlLacZ,respectively),theposi-tioningoflacZrelativetothesubgenomicpromoter,andtheefficiencyoftranslation.Wesuspectthatpromoter-strengthisthemajorfactor,butadditionalexperimentsarerequiredtotestthisidea.StudieswithotherDNA-basedSindbisvectorsalsosuggestthatpromoter-strengthinfluencesvectoreffi-ciency(17).

WealsoutilizedtheCMVIEpromotertogenerateahelperplasmid(pSCAHelper)inwhichexpressionofviralsPsisRNApolymeraseII-dependent.CotransfectionofpSCA␤andpSCA-Helpergeneratedvirusatatiterofaround106infectiouspar-ticles/ml(whichcouldbeartificiallylow,sinceX-galstainingwasusedtodeterminetiter,andthismethodhasrecentlybeenshowntounderestimatethenumberof␤-galactosidase-positivecells(20)).VirusgeneratedusingthenewDNAvectorsgivesthesamehighyieldofproteinasvirusgeneratedbytheorigi-nalRNA-basedapproach(50␮gof␤-galactosidase/106particlesofrecombinantvirus).

VirusstocksgeneratedusingpSCAHelperareconditionallyinfectiousduetoaminoacidchangeswithinsPp62atthejunctionbetweentheE2andE3spikeproteins(11).Thismod-ificationreducesthechanceofobtainingwild-typevirus,whichwouldrequirerecombinationbetweenre-RNAandhelperRNA,andreversionorsuppressionoftheconditionalmutation.Em-piricalmeasurementsshowthatthechanceofgeneratingRPV,followingtransfectionofre-RNAandhelperRNAthatencodes␣-chymotrypsin-activatedp62,islessthan10Ϫ10,andhasneverbeenobserved(11).AdisadvantageofDNAvectorsisthattheyprovideanotheropportunityforrecombination.ThelevelofRPVsobtainedfollowingtransfectionofSindbisDNA-basedrepliconandhelpervectorswas1000-foldhigher(ϳ107PFU/ml)thanthatobtainedaftertransfectionofinvitrogen-eratedre-RNAandhelperRNA(ϳ104PFU/ml)(17).Indeed,asimilarnumberofRPVsandrecombinantparticlesweregen-eratedusingtheDNA-basedsystemSindbisvectors.Incon-trast,noRPVsweredetectedinapopulationofnearly108recombinantparticlesgeneratedusingtheDNA-basedSFVsystemdescribedhere.ThesestatisticshighlightanimportantadvantageoftheSFVvectors.

Acknowledgments—WethankShi-YenMakforhelpinbuildingpSCA␤and⌬pSCA␤,andIrinaBurcescuforhelpinbuildingpSCA-Helper.WearealsoverygratefultoP.Liljestro¨mforprovidingpSFV1,pSFVHelper,andpSFV3-lacZ,andH.HerweijerandJ.A.WolffforprovidingpSin-nlLacZ.

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