Engineering the Japanese encephalitis virus RNA

Access Details:[subscription number 784374991]Publisher:Informa Healthcare

Informa Ltd Registered in England and Wales Registered Number: 1072954

Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Neurovirology

Publication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713669475Sang-Im Yuna;Yu-Jeong Choia;Xiao-Fang Yub;Jae-Young Songc;Young-HakShind;Young-Ran Jud;Seok-Yong Kima; Young-Min Leeaa

Department of Microbiology, College of Medicine and Medical Research Institute,Chungbuk National University, Cheongju, South Koreab

Department of Molecular Microbiology and Immunology, Bloomberg School ofPublic Health and Hygiene, Johns Hopkins University, Baltimore, Maryland, USAc

National Veterinary Research and Quarantine Service, Ministry of Agriculture andForestry, Anyang, South Korea

d

Division of Arboviruses, Center for Immunology and Pathology, Korea National Institute of Health, Seoul, South KoreaOnline Publication Date:01 November 2007

To cite this Article:Yun, Sang-Im,Choi, Yu-Jeong,Yu, Xiao-Fang,Song, Jae-Young,Shin, Young-Hak,Ju,

Young-Ran,Kim, Seok-Yong and Lee, Young-Min (2007) 'Engineering the Japanese encephalitis virus RNA genome forthe expression of foreign genes of various sizes: Implications for packaging capacity and RNA replication efficiency',Journal of Neurovirology, 13:6, 522 - 535

To link to this article: DOI:10.1080/13550280701684651URL:http://dx.doi.org/10.1080/13550280701684651Engineering the Japanese encephalitis virus RNAgenome for the expression of foreign genes of varioussizes: Implications for packaging capacity and RNAreplication efficiency

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use:http://www.informaworld.com/terms-and-conditions-of-access.pdfThisarticlemaybeusedforresearch,teachingandprivatestudypurposes.Anysubstantialorsystematicreproduction,re-distribution,re-selling,loanorsub-licensing,systematicsupplyordistributioninanyformtoanyoneisexpresslyforbidden.Thepublisherdoesnotgiveanywarrantyexpressorimpliedormakeanyrepresentationthatthecontentswillbecompleteoraccurateoruptodate.Theaccuracyofanyinstructions,formulaeanddrugdosesshouldbeindependentlyverifiedwithprimarysources.Thepublishershallnotbeliableforanyloss,actions,claims,proceedings,demandorcostsordamageswhatsoeverorhowsoevercausedarisingdirectlyorindirectlyinconnectionwithorarising out of the use of this material.Downloaded By: [McMaster University Library] At: 01:50 4 January 2008 JournalofNeuroVirology,13:522–535,2007󰀃c2007JournalofNeuroVirology

ISSN:1355-0284print/1538-2443onlineDOI:10.1080/13550280701684651

EngineeringtheJapaneseencephalitisvirusRNAgenomefortheexpressionofforeigngenesofvarioussizes:ImplicationsforpackagingcapacityandRNAreplicationefficiency

Sang-ImYun,1Yu-JeongChoi,1Xiao-FangYu,2Jae-YoungSong,3Young-HakShin,4Young-RanJu,4Seok-YongKim,1andYoung-MinLee1

ofMicrobiology,CollegeofMedicineandMedicalResearchInstitute,ChungbukNationalUniversity,

Cheongju,SouthKorea;2DepartmentofMolecularMicrobiologyandImmunology,BloombergSchoolofPublicHealthandHygiene,JohnsHopkinsUniversity,Baltimore,Maryland,USA;3NationalVeterinaryResearchandQuarantineService,MinistryofAgricultureandForestry,Anyang,SouthKorea;and4DivisionofArboviruses,CenterforImmunologyandPathology,KoreaNationalInstituteofHealth,Seoul,SouthKorea

UsingtheRNAreplicationmachineryofJapaneseencephalitisvirus(JEV),theauthorshaveestablishedandcharacterizedthreestrategiesfortheexpressionofforeigngenes.Initially,≈11kbgenomicRNAwasengineeredtoexpresshet-erologousgenesofvarioussizesbypreferentiallyinsertinganewcistronatthebeginningofthe3′nontranslatedvariableregion.RNAtransfectionyieldedrecombinantvirusesthatinitiatedforeigngeneexpressionafterinfectingper-missivecells.JEVwascapableofpackagingrecombinantgenomesaslargeas≈15kb.However,largergenomesizewasinverselycorrelatedwithRNAreplicationefficiencyandcytopathogenicity,withnosignificantchangeinin-fectivity.Second,avarietyofself-replicatingpropagation-deficientviralrepli-conswereconstructedbyintroducingonetothreein-framedeletionsintotheectodomainsofallthestructuralproteinsofJEV.TheserepliconsdisplayedaspectrumofRNAreplicationefficiencyupontransfection,suggestingthatrem-nanttransmembranedomainsplayasuppressiveroleinthisprocess.Third,theauthorsgeneratedapanelofstablepackagingcelllines(PCLs)providingallthreeJEVstructuralproteinsintrans.ThesePCLsefficientlypackagedviralrepliconRNAsintosingle-roundinfectiousviralrepliconparticles.TheseJEV-basedvirus/vectorsystemsmayprovideusefultoolsforavarietyofbiologicalapplications,includingforeigngeneexpression,antiviralcompoundscreen-ing,andgeneticimmunization.JournalofNeuroVirology(2007)13,522–535.

1Department

Keywords:encapsidation;foreigngeneexpression;Japaneseencephalitis

virus;packagingcelllines;trans-complementation;viralrepliconparticles

Introduction

Japaneseencephalitisvirus(JEV),amosquito-borneflavivirus,iscloselyrelatedtodenguevirus(DENV),yellowfevervirus(YFV),WestNilevirus(WNV),andKunjinvirus(KUNV).Likeotherflaviviruses,JEVisasmall-envelopedviruswithasingle-strandedpositive-senseRNAgenomeof≈11,000nucleotides(nt)inlength,whichiscappedatthe5′endandunpolyadenylatedatthe3′end(LindenbachandRice,2001).Thegenomecontainsasinglelongopenreadingframe(ORF)flanked

AddresscorrespondencetoYoung-MinLee,DepartmentofMi-crobiology,CollegeofMedicineandMedicalResearchInstitute,ChungbukNationalUniversity,12Gaeshin-Dong,Heungduk-Ku,Cheongju-Si,SouthKorea.E-mail:ymlee@chungbuk.ac.kr

Supplementalmaterialsforthisarticleareprovidedseparately.ThisworkwassupportedbyaKoreaResearchFoundationgrant(KRF-2001-042-D00071),RepublicofKorea.TheauthorsthankDr.DeborahMcClellanforeditorialsupport.

Received11May2007;revised11July2007;accepted9August2007.

Library] At: 01:50 4 January 2008 JEV-basedforeigngenedeliveryS-IYunetal

523

bycis-actingnontranslatedregions(NTRs)forvi-ralreplication/transcription/translationatboththe5′and3′ends,whicharecharacterizedbyhighlycon-servedprimarysequencesandsecondaryortertiarystructures(Markoff,2003).Thepolyproteinisco-orpost-translationallyprocessedintomatureproteinsbycellularandviralproteases.TheinfectiousvirionisassembledbyencapsidatinggenomicRNAintothecoreshellofcapsid(C)proteins,whichis,inturn,envelopedbytwoviralglycoproteins,premembrane2005;Bredenbeeketal,2006;Jonesetal,2005;LaiandMonath,2003;Molenkampetal,2003),JEVhasnotbeenusedforthispurposetodate.Herewere-portthreestrategiesforutilizingJEVoritsgeneticelementsasvectorstoexpressforeigngenesinava-rietyofcelltypes,takingadvantageofafull-lengthinfectiousJEVcDNA(Yunetal,2003)thatwehavepreviouslyconstructed.TheseJEV-basedexpressionsystemscanpotentiallyserveaspowerfultoolsinbothinvitroandinvivoapplications.Thelargepack-yt(prM;whichisfurtherprocessedintotheprandMisrproteins)andenvelope(E),embeddedinthehost-evderivedlipidmembrane.TheRNAgenomereplicatesinUinthecytoplasm.Thisprocessismediatedbyacom- rplexofviralreplicases,includingNS1,NS2A,NS2B,etsNS3,NS4A,NS4B,andNS5(LindenbachandRice,aM2001).

cSeveraloftheflaviviruses,includingJEV,displayaM[numberofcharacteristicsthatmakethemusefulfor :ythedevelopmentofdeliveryvectorstoexpressfor-B eigngenes:Severalflavivirushostsaresusceptibledetoinfection,andawiderangeofcelltypes,includ-daingthoseofinsect,avian,andmammalianorigin,olnarepermissiveforinfectionandreplication.Also,wmanipulatablegenomesizesandsimpleproceduresoDmakepossibletherapidgenerationofhightitersofrecombinantviruses.Inaddition,cytoplasmicRNAamplificationintheseviruseseliminatesnuclearin-volvementandleadstohighlevelsofreplicationandgeneexpression.Inthepastfewyears,heterologousgeneshavebeenexpressedinthecontextof(i)aninfectiousvirusgenome,inthecaseofWNV(Pier-sonetal,2005)andtick-borneencenphalitisvirus(TBEV)(Gehrkeetal,2005);or(ii)arepliconlack-ingviralstructuralproteins(C,prM,andE),inthecaseofseveralflaviviruses,includingDENV(Pangetal,2001),YFV(Jonesetal,2005;Masonetal,2006;Molenkampetal,2003),WNV(Fayzulinetal,2006;Loetal,2003;Masonetal,2006;Shietal,2002),KUNV(KhromykhandWestaway,1997;VarnavskiandKhromykh,1999),andTBEV(Gehrkeetal,2005).InthecaseofYFV,twoapproachestoproducingre-combinantvirusesusinginfectiouscDNAtechnol-ogyhavebeenutilized,dependingontheantigentobeexpressed.OneisthegenerationofchimericvirusesthroughtheexchangeofstructuralprM/Egenes(LaiandMonath,2003).Theothertechniqueistheexpressionofforeignproteinepitopesatthesurfaceofrecombinantvirusesbyengineeringcer-tainEprotein–codingregions(Bonaldoetal,2002;Bonaldoetal,2005).

JEVisoneofonlytwoflaviviruses(theotherbe-ingYFV17D)forwhichaneffectiveliveattenuatedvaccine(SA14-14-2forJEV)isavailableforhumanvaccination(Hennessyetal,1996;Monath,2003;Solomon,2003;Xinetal,1988).Consequently,itisaparticularlyattractivecandidateforthedevelopmentofgeneexpressionsystems.However,incontrasttoYFV,whichhasbeenextensivelyexploitedasavec-torforexpressingforeigngenes(Bonaldoetal,2002,

agingcapacityofJEVoffersadistinctadvantagewithregardtoexpressinglargergenesandthetreatmentofassociateddiseases.Ourfindingsalsoindicatethatcross-talkmaylinkthebiogenesisoftheJEVstruc-turalproteinswithintheERtothelevelofRNArepli-cationinthecytoplasmofinfectedcells.

Results

RecombinantJEVgenomicRNAsofupto≈15kbareencapsidatedintoinfectiousvirionswithnosignificantchangesininfectivity,butlargergenomesizeisinverselycorrelatedwithRNAreplicationefficiencyandcytopathogenicityincellculture

Todetermineanappropriatesitefortheinsertionofanadditionalforeigngeneexpressionunitwithinthefull-lengthgenomicRNAofJEV,weinitiallycon-structedapairofrecombinantfull-lengthJEVcDNAsbyinsertinganexpressioncassetteforanenhancedAequoreavictoriagfp(egfp)geneasareporterattwodifferentlociwithintheviralgenomewithoutdis-ruptingitssinglelongORF,eitherupstreamoftheinitiationcodonordownstreamofthestopcodon,inwhatisknownasthevariableregionofJEV3′NTR(detaileddescriptiongiveninFigureS1,Sup-plementalMaterials).DirectcomparisonofthesetworecombinantcDNAsrevealedthattheinsertionofthehighlystructuredinternalribosomeentrysiteoftheencephalomyocarditisvirus(EMCVIRES)RNAse-quenceandthe768-bpegfpgeneatthevariablere-gionoftheJEV3′NTRallowedthecompleterepli-cationofJEVbutreducedRNAproductionandviralproteinexpression(FiguresS1andFigures1to3).Incontrast,insertionattheJEV5′NTRupstreamoftheinitiationcodonofthesinglelongORFhadadelete-riouseffectonreplication(FigureS1).

Next,wecharacterizedthepackagingcapacityofJEVandevaluateditsusefulnessforexpressingfor-eigngenes,usingapanelofrecombinantJEVge-nomicRNAsofvariablesizes.BasedonastrategyofinsertinganadditionalEMCVIRES-drivenfor-eigngene′expressionunitatthevariableregionoftheJEV3NTR,weengineeredthefull-lengthvi-ralgenometoexpressthreecommonlyusedandvariouslysizedheterologousreportergenes:egfp(768bp),theluciferasegenefromPhotinuspyralis(luc,1653bp),andlacZ(3012bp).TheresultingthreerecombinantcDNAconstructsweredesignatedpJEV/FL/3egfp,pJEV/FL/3luc,andpJEV/FL/3lacZ,respectively(Figure1).Inparallel,weconstructed

Downloaded By: [McMaster University Library] At: 01:50 4 January 2008 524

JEV-basedforeigngenedelivery

S-IYunetal

Figure1PackagingofrecombinantJEVgenomicRNAsofupto≈15kbinsizewithnosignificantchangeininfectivityorproductionofrecombinantviruses.AnewcistrondrivenbyEMCVIRESfortheexpressionofthreereportergenesofdifferentsizes(egfp,768bp;luc,1653bp;lacZ,3012bp)wasinsertedintothevariableregionofJEV3′NTRinpJEV/FL,producingthreerecombinantcDNAs(pJEV/FL/3egfp,pJEV/FL/3luc,andpJEV/FL/3lacZ).ThestructuresoftherecombinantJEVcDNAsandparentalcDNA(pJEV/FL)areshown.ViralORFsareillustratedbythicksolidlinesatbothtermini,whichdenotethe5′and3′NTRsofthegenome.Inthecaseofreplication-competentpJEV/FL/3luccDNA,replication-incompetentpJEV/FL/3lucREP−wasalsogeneratedasanegativecontrolbyintroducingan83-ntinternaldeletion(dottedline)inthemiddleoftheNS3gene,resultingintheprematureterminationofviraltranslationatnt5596(asterisk).SP6polymeraserunofftranscriptionofrecombinantJEVcDNAtemplatesproducedgenomicRNAsranginginsizefrom10,968to14,753nt(viralgenomesize).Na¨ıveBHK-21cells(8×106)weretransfectedwith2µgofsyntheticRNAstranscribedfromeachcDNAtemplate.ThespecificinfectivitiesofthesyntheticRNAswereestimatedbycountingtheplaque-formingunits(PFU)perµgofRNA,thegreenfocus-formingunits(GFU)perµgofRNAforpJEV/FL/3egfpunderafluorescencemicroscope,orthebluefocus-formingunits(BFU)perµgofRNAforpJEV/FL/3lacZafterX-galstaining,asindicated(infectivity).Virustitersintheculturesupernatantsweredeterminedat48and72hposttransfectionbymeasuringthenumbersofPFUperml,GFUpermlforpJEV/FL/3egfp,orBFUpermlforpJEV/FL/3lacZ,asindicated(virustiter).h.p.t.,hoursposttransfection.

thereplication-incompetentpJEV/FL/3lucREP−(Yunetal,2003)asanegativecontrol;thisconstructcon-tainsaninternaldeletionof83ntintheNS3regionthatprematurelyterminatesviraltranslationatnt5596(Figure1).FromthesefiverecombinantcDNAs,includingparentalpJEV/FL,fiverecombinantJEVgenomicRNAsofvariouslengths(10,968to14,753nt)weresynthesizedbyinvitrorun-offtranscriptionusingSP6RNApolymerase(Figure1,viralgenomesize).

UsingthesenewlygeneratedRNAs,weinitiallysoughttoidentifythemechanismbywhichthegenomesizeofJEVmightinfluencethespecificinfectivityandreplicationoftheviralgenomicRNA,expressionofviralproteins,andproduc-tionofinfectiousviralparticles.WefoundthatthesyntheticRNAsderivedfrompJEV/FL/3egfp,pJEV/FL/3luc,andpJEV/FL/3lacZ,whenintroducedintosusceptibleBHK-21cells,displayedspecificin-fectivitiesof1.1–2.0×106,0.8–0.9×106,and0.6–0.8×106plagueformingunits(PFU)/µg,respec-tively,analagoustothatofsyntheticRNAde-rivedfromparentalpJEV/FL(1.6–2.2×106PFU/µg)(Figure1,infectivity).Wealsosawnoevi-denceofdifferencesinspecificinfectivityby

confocalmicroscopyforpJEV/FL/3egfp-derivedorpJEV/FL/3lacZ-derivedRNA-transfectedcells.How-ever,weobservedadelayintheaccumulationofin-fectiousviralparticlesinsupernatantfractionsfromrecombinantRNA-transfectedBHK-21cells,andthisdelaywasdirectlycorrelatedwiththelengthsoftheheterologousreportergenesinserted(Figure1,virustiter).Thiscorrelationwasreflectedbythefor-mationofsmallerplaques(Figure2a),delayedac-cumulationofgenomicRNAsasmeasuredbyreal-timequantitativereversetranscription–polymerasechainreaction(RT–PCR)(Figure2b),anddelayedexpressionofviralproteinsasvisualizedbyim-munoblottingwithaJEV-specifichyperimmuneanti-serum(Figure2c)inrecombinantRNA-transfectedBHK-21cells.Thus,JEVwascapableofencapsi-datingrecombinantgenomesaslargeas≈15kb,butthepackagingoftheselargergenomeswasneg-ativelycorrelatedwithRNAreplicationefficiencyandcytopathogenicityincellcultureandnotwithinfectivity.

WethenexaminedthefunctionalintegrityoftherecombinantRNAsbyanalyzingtheexpres-sionofvariouslengthsofheterologousreportergenesinsertedintotheviralgenome(Figure3).

Downloaded By: [McMaster University Library] At: 01:50 4 January 2008 JEV-basedforeigngenedeliveryS-IYunetal

525

Figure2NegativecorrelationoftheJEVgenomicRNAsizewiththelevelofRNAreplicationandextentofcytopathogenicity.Na¨ıveBHK-21cells(8×106)weremock-transfectedortransfectedwith2µgoftheparentorrecombinantJEVRNAstranscribedfromtherelevantJEVcDNA,asindicated.(a)Representativeplaquemorphology.Transfectedcellswereoverlaidwithagaroseandstained5dayslaterwithcrystalviolet.(b)LevelofJEVRNAproduction.TotalcellularRNAwasisolatedfromtransfectedcellsattheindicatedtimepoints.Real-timequantitativeRT–PCRwasusedforthequantitationofJEV-specificRNAandβ-actinRNAfornormalizationoftotalRNAlevels.The2−󰀄󰀄CTmethodwasusedtoanalyzethechangesinJEVRNAlevels,relativetothoseat6hposttransfection,obtainedfromreal-timequantitativePCRexperiments.Dataareshownfromoneoftwoindependentexperiments,whichyieldedsimilarresults.(c)LevelsofJEVproteinaccumulation.Transfectedcellswerelysedwith1×sampleloadingbufferattheindicatedtimepoints,andtheproteinextractswereresolvedon10%SDS-polyacrylamidegels.ViralproteinswerevisualizedbyimmunoblottingwithaJEV-specifichyperimmuneantiserum.Thepositionsoftheviralproteins(E,NS1,andNS3)andacleavage-relatedintermediate(arrowhead)areindicatedontheleft.MolecularmassmarkersinkDaarespecifiedontheright.*and**indicatethat>50%and>99%ofthepJEV/FL-derivedRNA-transfectedcellsdiedanddetachedfromthebottomoftheculturedishes,respectively.V,JEVCNU/LP2-infectedBHK-21cellsasareference;N,na¨ıveBHK-21cells.h.p.t.,hoursposttransfection.

InpJEV/FL/3egfp-derivedRNA-transfectedBHK-21cells,confocalmicroscopydisclosedgreenfluores-cenceinboththenucleusandcytoplasmofcellsex-pressingEGFP(Figure3a),becauseenhancedgreenfluorescentprotein(EGFP)issmall(≈30kDa)enoughtodiffusebetweenthenucleusandcytoplasm.Asexpected,fluorescencewasnotobservedinmock-transfectedcells(Figure3a).Flowcytometryanalysesdemonstratedthatgreenfluorescentcellsconstituted99.8%ofthetransfectedcells,whencomparedtomock-transfectedcells(Figure3b).InpJEV/FL/3lacZ-derivedRNA-transfectedBHK-21cells,theX-galstainingpatternoflacZ-expressingcells,asrevealedbylightmicroscopy,indicatedthat99.6%ofthetransfectedcellsexpressedlacZ(Figure3c).WealsomonitoredtheluciferaseactivityofBHK-21cellsovertimeaftertransfectionwitheitherthereplication-competentpJEV/FL/3luc-derivedRNAorwithreplication-incompetentpJEV/FL/3lucREP−-

derivedRNAasanegativecontrol(Figure3d).Wefoundthatthelevelofluciferaseactivityvariedovertime,dependingonthepresenceorabsenceofviralreplication.

Self-replicating,self-limitingJEVrepliconsconstructedbydeletingone,two,orallviralstructuralgenesdisplayaspectrumofRNAreplicationandproteinexpression

ToindependentlyexpressforeigngenesusingtheJEVRNAreplicationmachinery,weusedpJEV/FL/3luctogenerateapanelofself-replicating,self-limitingvi-ralrepliconsthatmeetstringentsafetyconcerns.Weemployedthelucreporterastheheterologousgenebecauseitallowssensitiveandquantitativemon-itoringofviralreplication.Inall,weconstructedthreesetsofnineviralrepliconvectorsbyintro-ducingonetothreein-framedeletionsintotheectodomainsofalltheJEVstructuralproteinsin

Downloaded By: [McMaster University Library] At: 01:50 4 January 2008 526

JEV-basedforeigngenedelivery

S-IYunetal

Figure3FunctionalassaysfortheexpressionofthreereportergenesencodedineachofthethreerecombinantJEVgenomicRNAsofvariablesizes.NaiveBHK-21cells(8×106)weremock-transfectedortransfectedwith2µgoftherecombinantJEVRNAstranscribedfromeachcDNA,asindicated:pJEV/FL/3egfp(a,b),pJEV/FL/3lacZ(c),pJEV/FL/3lucorpJEV/FL/3lucREP−(d).(a,b)Expressionofegfp.Transfectedcellswerepreparedforconfocalmicroscopy(a)andflowcytometricanalysis(b).dottedline,mock-transfectedcells;solidline,cellstransfectedwithpJEV/FL/3egfp-derivedRNA.(c)ExpressionoflacZ.TransfectedcellswereprocessedforX-galstaining.(d)Expressionofluc.Transfectedcellswereseededon6-wellplatesatadensityof6×105cellsperwell.Attheindicatedtimepoints,celllysateswereassayedforluciferaseactivity.Experimentswereperformedintriplicate.Dataarepresentedasmeanvalues,withthestandarddeviationsindicatedbyerrorbarssolidcircles,pJEV/FL/3luc-derivedRNA-transfectedcells;opencircles,pJEV/FL/3lucREP−-derivedRNA-transfectedcells;—,thelevelofbackgroundluminescenceofna¨ıveBHK-21cells.h.p.t.,hoursposttransfection.

ordertoallowustocomparetheinfluenceofeachdeletiononthecompetenceandlevelofRNArepli-cation:(i)afirstsetoffour(pJEV/Rep/3luc/󰀄CC,pJEV/Rep/3luc/󰀄C,pJEV/Rep/3luc/󰀄prM,andpJEV-/Rep/3luc/󰀄E)bearingasingledeletionineachoftheviralstructuralprotein-codingregions;(ii)asec-ondsetofthree(pJEV/Rep/3luc/󰀄C+󰀄prM,pJEV/Rep/3luc/󰀄C+󰀄E,andpJEV/Rep/3luc/󰀄prM+󰀄E)containingadoubledeletioninthecodingre-gionsofparticularstructuralproteins;and(iii)athirdsetoftwo(pJEV/Rep/3luc/󰀄C+󰀄prM+󰀄EandpJEV/Rep/3luc/󰀄CtoE)lackingallthestructuralpro-teins(Figure4).Inallcases,theregionofdeletionwascarefullydeterminedwithrespecttothemem-branetopologyofeachstructuralproteinsothatthetransmembranedomainslocatedintheC-terminire-mainedintact.pJEV/Rep/3luc/󰀄CCwasidenticaltopJEV/Rep/3luc/󰀄C,exceptthatanadditional5′dele-tion(nt132to201)extendedtoincludethepro-posedcyclizationsequencemotifinthe5′regionoftheCgene,whichisrequiredforreplicationinothermosquito-borneflaviviruses,suchasDENV,YFV,KUNV,andWNV(Alvarezetal,2005;Breden-beeketal,2003;Corveretal,2003;Hahnetal,1987;Khromykhetal,2001;Loetal,2003;Youetal,2001).WethenaskedwhethertheviralrepliconRNAsderivedfromninecDNAtemplateswerecom-petentinreplicationbyanalyzingviralpro-teinexpressionovertimeaftertransfectionintoBHK-21cells.TwoRNAsamplesderivedfromreplication-competentpJEV/FL/3lucandreplication-incompetentpJEV/FL/3lucREP−weretransfectedinparalleltoserveaspositiveandnegativecontrols,respectively.Immunoblottingusingananti-JEVNS1antiserum(Figure5a)andaJEV-specifichyperim-muneantiserum(datanotshown)showedthatwiththeexceptionofpJEV/Rep/3luc/󰀄CC-derivedRNA,allviralrepliconRNAsharboringasingle,dou-ble,ortriplein-framedeletioninJEVstructuralgenesproducedadetectableamountofviralpro-teinsduringthefirst24hposttransfection.Pro-teinlevelsincreaseduntil48hposttransfectionandweremaintaineduntil72hposttransfectionbecauseofalackofcell-to-cellspread.Incontrast,wedidnotdetectanyviralproteinsinBHK-21cellstransfectedwithpJEV/Rep/3luc/󰀄CC-derivedRNAorwithreplication-incompetentpJEV/FL/3lucREP−-derivedRNA(Figure5aanddatanotshown).Asexpected,inBHK-21cellstransfectedwithreplication-competentpJEV/FL/3luc-derivedRNA,

Downloaded By: [McMaster University Library] At: 01:50 4 January 2008 JEV-basedforeigngenedeliveryS-IYunetal

527

Figure4SchematicpresentationofJEVreplicons.AllJEVrepliconsdesignedtoexpressthelucgenewereconstructedonthebasisofpJEV/FL/3lucbyintroducingone,two,orthreeinternalin-framedeletions(dottedlines)intheectodomain-codingregionsofallthreeviralstructuralproteins(C,prM,andE).Thefirstsetoffourreplicons(pJEV/Rep/3luc/󰀄CC,pJEV/Rep/3luc/󰀄C,pJEV/Rep/3luc/󰀄prM,andpJEV/Rep/3luc/󰀄E)containedasingleinternalin-framedeletionineachstructuralgeneofJEV.pJEV/Rep/3luc/󰀄CCwasidenticaltopJEV/Rep/3luc/󰀄C,exceptforanadditional5′deletion(nt132-201)thatextendedtotheproposedcyclizationsequencemotifinthe5′re-gionoftheCgene.Thesecondsetofthreereplicons(pJEV/Rep/3luc/󰀄C+󰀄prM,pJEV/Rep/3luc/󰀄C+󰀄E,andpJEV/Rep/3luc/󰀄prM+󰀄E)containedtwointernalin-framedeletionsinthestructuralgenes.Thethirdsetoftworeplicons(pJEV/Rep/3luc/󰀄C+󰀄prM+󰀄E,andpJEV/Rep/3luc/󰀄CtoE)lackedallthreeJEVstructuralproteins.pJEV/Rep/3luc/󰀄C+󰀄prM+󰀄Econtainedthreeinternalin-framedele-tionsintroducedintoeachofthethreestructuralgenes,whereaspJEV/Rep/3luc/󰀄CtoEcontainedthe35N-terminaland24C-terminalaminoacidsoftheCprotein,immediatelyfollowedbytheN-terminusoftheNS1proteinandtherestoftheviralgenome.OneortwotransmembranedomainslocatedattheC-terminusofeachstructuralprotein(oneforCandtwoforprMandE)arerepresentedbyblackverticalbars.Thelocus,size,andpositionoftheinternalin-framedeletionsintroducedintheectodomain-codingregionsofallthreeviralstructuralproteinsareshownontheright.

Downloaded By: [McMaster University Library] At: 01:50 4 January 2008 528

JEV-basedforeigngenedelivery

S-IYunetal

Figure5ThespectrumofreplicationefficienciesexhibitedbyapanelofJEVrepliconRNAs.Na¨ıveBHK-21cells(8×106)weremock-transfectedortransfectedwith2µgoftheparentorJEVrepliconRNAstranscribedfromeachcDNA,asindicated.M,mock;WT,pJEV/FL/3luc;REP-,pJEV/FL/3lucREP−;󰀄CC,pJEV/Rep/3luc/󰀄CC;󰀄C,pJEV/Rep/3luc/󰀄C;󰀄prM,pJEV/Rep/3luc/󰀄prM;󰀄E,pJEV/Rep/3luc/󰀄E;󰀄C+󰀄prM,pJEV/Rep/3luc/󰀄C+󰀄prM;󰀄C+󰀄E,pJEV/Rep/3luc/󰀄C+󰀄E;󰀄prM+󰀄E,pJEV/Rep/3luc/󰀄prM+󰀄E;󰀄C+󰀄prM+󰀄E,pJEV/Rep/3luc/󰀄C+󰀄prM+󰀄E;󰀄CtoE,pJEV/Rep/3luc/󰀄CtoE.h.p.t,hourspost-transfection.(a)LevelsofJEVNS1proteinaccumulationovertimepost-transfection.Transfectedcellswerelysedwith1×sampleloadingbufferattheindicatedtimepoints,andtheproteinextractswereresolvedon10%SDS-polyacrylamidegels.Viralproteinswerevisualizedbyimmunoblottingwithananti-JEVNS1rabbitantiserum.(b–d)LevelsofJEVRNAproduction(b),JEVNS1proteinaccumulation(c),andluciferaseactivity(d)at48hposttransfection.(b)Real-timequantitativeRT-PCRexperiments.TransfectedcellswereusedfortheisolationoftotalcellularRNAattheindicatedtimepoints.Real-timequantitativeRT-PCRwasemployedforthequantitationofJEV-specificRNAandβ-actinRNAfornormalizationoftotalRNAlevels.Weutilizedthe2−󰀄󰀄CTmethodtoanalyzethechangesinJEVRNAlevels,relativetothoseat6hposttransfectionfromreal-timequantitativePCRexperiments.Dataareshownfromoneoftwoindependentexperiments,whichyieldedsimilarresults.(c)Westernblotanalyses.Equalamountsofproteinextractsfromtransfectedcellsat48hposttransfectionwereseparatedon10%SDS-polyacrylamidegels.TheJEVNS1proteinwasvisualizedbyimmunoblottingwithananti-JEVNS1rabbitantiserum.(d)Luciferaseassays.Equalamountsofcelllysatesfromtransfectedcellsat48hposttransfectionwereusedtodetermineluciferaseactivity.Experimentswereperformedintriplicate,anddataarepresentedasmeanvalueswiththestandarddeviations,indicatedbyerrorbars.

JEVNS1(Figure5a)andotherJEV-specificviralproteins(datanotshown)weredetectableat24hposttransfectionandgraduallyaccumulateduntil72hposttransfection.Thus,withtheexceptionofthepJEV/Rep/3luc/󰀄CC-derivedRNA,allthevi-ralrepliconRNAsharboringasingle,double,ortriplein-framedeletioninJEVstructuralgeneswerereplication-competent.

However,differencesbecameevidentbetween48and72hposttransfection(Figure5a).Inrepeatedexperiments,directcomparisonofthelevelsofviralrepliconRNAsbyreal-timequan-titativeRT–PCRshowedthatproductionofthepJEV/Rep/3luc/󰀄E-derivedRNAwasconsistentlyhigherthanthatbyallotherviralrepliconRNAsharboringasingle,double,ortriplein-framedele-tioninJEVstructuralgenes(Figure5b).At48hposttransfection,productionofthetwoviralrepli-conRNAsderivedfromeitherpJEV/Rep/3luc/󰀄CorpJEV/Rep/3luc/󰀄prMwas≈5.7-to7.2-foldlowerthanthatofpJEV/Rep/3luc/󰀄E-derivedRNA.Also,productionofthetwoviralrepliconRNAsderivedfromeitherpJEV/Rep/3luc/󰀄C+󰀄EorpJEV/Rep/3-luc/󰀄prM+󰀄Ewas≈1.2-foldlower,andthatofpJEV/Rep/3luc/󰀄C+󰀄prM+󰀄E-derivedRNAwas≈10.6-foldlowerthanthatofpJEV/Rep/3luc/󰀄E-derivedRNA.Inaddition,productionofpJEV/Rep/3luc/󰀄C+󰀄prM-derivedRNAwas≈1.4-to1.8-foldlowerthanthatofthetwoviralrepliconRNAsderivedfromeitherpJEV/Rep/3luc/󰀄CorpJEV/Rep/3luc/󰀄prMat48hpost-transfection.Inallcases,thespectrumofRNAproductionclearlycor-relatedwiththeaccumulationlevelsoftheJEVNS1protein(Figure5c)andtheexpressionprofilesoftheLUCprotein(Figure5d).

Thus,twoin-framedeletionsintroducedintheectodomainsoftheCandprMproteins,individ-uallyorincombination,inthecontextofeither

Downloaded By: [McMaster University Library] At: 01:50 4 January 2008 JEV-basedforeigngenedeliveryS-IYunetal

529

Figure6ConstructionandcharacterizationofthepackagingsystemforJEVrepliconRNAs.(a)SchematicpresentationofthreeJEVstructuralproteinexpressionvectorsbasedontheSindbisvirus–derivedexpressionvector,pSinRep19.Aforeigngeneandthepacgenewereexpressedusingseparatesubgenomicpromoters(26Spromoter),indicatedbyarrows.ThepSinRep19/JEVC-EvectorcontainstheentirecodingsequenceofJEVCthroughE.ThepSinRep19/JEVC-E-BglIIvectorincludesthecompletecodingsequenceofJEVCthroughE,followedbytheN-terminal58residuesofNS1.ThepSinRep19/JEVC-NS1encompassesthecompletecodingsequenceofJEVCthroughNS1.MCS,multiplecloningsites.(b)ExpressionofJEVstructuralproteins.Na¨ıveBHK-21cellsweremock-transfectedortransfectedwithRNAtranscriptsderivedfromeachJEVstructuralproteinexpressionvector,asindicated.Celllysateswereharvested48hlater.EqualamountsofcelllysateswereresolvedbySDS-PAGEandprobedwithaJEV-specifichyperimmuneantiserum(toppanel).Inparallel,GAPDHproteinwasdetectedasaloadingandtransfercontrolwithananti-GAPDHrabbitantiserum(bottompanel).ThepositionsofviralproteinsEandNS1arespecifiedontheright,andthemolecularmassmarkersinkDaontheleft.

pJEV/Rep/3luc/󰀄EorparentalpJEV/FL/3luc,actedsynergisticallyinsuppressingRNAproduction.Thisfindingsuggestedthatexpressionofonlythetrans-membranedomainsofJEVstructuralproteins,intheabsenceoffunctionalproteins,mightsuppressge-nomicRNAreplication.Thispossibilitywassup-portedbyadirectcomparisonofpJEV/Rep/3luc/󰀄C+󰀄prM+󰀄EtopJEV/Rep/3luc/󰀄CtoE,whichcontainsthe35N-terminaland24C-terminalaminoacidsoftheCprotein,immediatelyfollowedbytheN-terminusoftheNS1proteinandtherestoftheviralgenome(Figure4).RNAproductionbypJEV/Rep/3luc/󰀄CtoEwas≈6.5-foldhigherthanthatbypJEV/Rep/3luc/󰀄C+󰀄prM+󰀄Eat48hpost-transfection(Figure5b),indicatingthattheremovaloftwopairsoftransmembranedomains(oneattheendoftheprMandtheotherattheendoftheE)inpJEV/Rep/3luc/󰀄C+󰀄prM+󰀄EincreasedtheRNAreplicationefficiency.Consistently,RNAlevelswerereflectedintheaccumulationoftheJEVNS1protein(Figure5c)andtheexpressionprofilesofLUCprotein(Figure5d).

GenerationofstablepackagingcelllinescapableofencapsidatingJEV-derivedrepliconvectorRNAsintosingle-roundinfectiousviralrepliconparticlesTheutilityofJEVreplicon-basedexpressionvectorswasexpandedbydevelopingatrans-complementationsystemthatexpressedallthreeJEVstructuralproteinsintransandallowedtheen-

capsidationofJEVrepliconRNAsintosingle-roundinfectious,propagation-deficientviralrepliconparti-cles(VRPs).FortheectopicexpressionofJEVstruc-turalproteins,weutilizedaSindbisvirus–basedheterologousgeneexpressionvector,pSinRep19(Agapovetal,1998),thatcontainsthepuromycinN-acetyltransferase(pac)geneasadominantse-lectablemarker.WeconstructedasetofthreeJEVstructuralproteinexpressionvectorscontainingtheentirecodingsequenceofJEVCthroughE(pSinRep19/JEVC-E),JEVCthroughEplusthe58N-terminalresiduesofNS1(pSinRep19/JEVC-E-BglII),andJEVCthroughNS1(pSinRep19/JEVC-NS1)(Figure6a).ProteinexpressionwasevaluatedinBHK-21cellsaftertransfectionwithsyntheticRNAstranscribedinvitrofromthecorrespondingvector.ImmunoblotanalysisoflysatesfromtransfectedcellswithaJEV-specifichyperimmuneantiserumrevealedthatequalamountsoftheJEVEproteinwereexpressedincellstransfectedwithoneofthethreevectorRNAs,whereasJEVNS1wasidentifiedonlyinBHK-21cellstransfectedwiththevectorRNAtranscribedfrompSinRep19/JEVC-NS1(Fig-ure6b).GAPDHwasemployedasaloadingcontrol(Figure6b).

WetooktwoapproachestoproducingJEVVRPs(Figure7a).Thefirstinvolvedthetransientco-transfectionofJEVrepliconRNAwithoneofthethreeJEVstructuralproteinexpressionvectorRNAs.TiteringandmonitoringofVRPproduction

Downloaded By: [McMaster University Library] At: 01:50 4 January 2008 530

JEV-basedforeigngenedelivery

S-IYunetal

Figure7Productionofsingle-roundinfectiouspropagation-deficientJEVVRPs.(a)SchematicillustratingthegenerationofJEVVRPsby(i)cotransfectionofJEVstructuralproteinexpressionvectorRNAswithJEVrepliconRNAsor(ii)transfec-tionofJEVstructuralprotein-expressingPCLswithJEVrepliconRNAs.(b,c)ProductionofJEVVRPs.Twoapproacheswereutilized:Oneinvolvedthetransientcotransfectionofna¨ıveBHK-21cellswithtwovectorRNAs,includingaJEVstructuralproteinexpressionvectorRNAalongwithaJEVrepliconRNA,asindicated(b).Theotherprocedurein-volvedthetransfectionofJEVPCLswithoneoftheJEVrepliconRNAs(c).TheJEVrepliconRNAsusedwereasfol-lows:󰀁green,pJEV/Rep/3egfp/󰀄C+󰀄prM+󰀄E;󰀂green,pJEV/Rep/3egfp/󰀄CtoE;󰀁blue,pJEV/Rep/3lacZ/󰀄C+󰀄prM+󰀄E;󰀂blue,pJEV/Rep/3lacZ/󰀄CtoE;󰀁black,pJEV/Rep/3luc/󰀄C+󰀄prM+󰀄E;󰀂black,pJEV/Rep/3luc/󰀄CtoE.Thesupernatantfractionswerecol-lectedat48hposttransfectionandusedtoinfectna¨ıveBHK-21cellsforthetitrationofVRPs,followedbytheexaminationoftherespectivereportergeneexpression.−,thelevelofbackgroundluminescenceofna¨ıveBHK-21cellsforluciferaseactivity.

wasperformedbyinfectingna¨ıveBHK-21cellsandassayingforreportergeneexpressionfromtheJEVrepliconRNApackagedintoVRPs.Co-transfectionofpSinRep19/JEVC-NS1-derivedvec-torRNAwithegfp-expressingJEVrepliconRNAde-rivedfromeitherpJEV/Rep/3egfp/󰀄C+󰀄prM+󰀄EorpJEV/Rep/3egfp/󰀄CtoEinseveralexperimentspro-duced1.1–4.3×104infectiousunits/ml(IU/ml)ofVRPs(Figure7b).Similarresultswereob-tainedusinglacZ-expressingJEVrepliconRNAde-rivedfromeitherpJEV/Rep/3lacZ/󰀄C+󰀄prM+󰀄EorpJEV/Rep/3lacZ/󰀄CtoE(Figure7b).NoapparentdifferenceswereevidentwhenpSinRep19/JEVC-E-BglII–derivedvectorRNAwasusedfortheexpres-sionofJEVstructuralproteinsinsteadofthatfrompSinRep19/JEVC-NS1(Figure7b).Incontrast,co-transfectionofthepSinRep19/JEVC-E-derivedvectorRNAwithoneoffourJEVrepliconRNAsexpress-ingeitheregfporlacZconsistentlyproduced≈100-foldfewerVRPs(Figure7b).TheseobservationswereconfirmedbycotransfectingoneofthreepSinRep19-basedJEVstructuralproteinexpressionvectorRNAswithoneoftwoluc-expressingJEVrepliconRNAsderivedfromeitherpJEV/Rep/3luc/󰀄C+󰀄prM+󰀄EorpJEV/Rep/3luc/󰀄CtoE(Figure7b).

ThesecondapproachtoproducingJEVVRPswasbasedonusingstablepackagingcelllines(PCLs),es-tablishedbyinitiallytransfectingna¨ıveBHK-21cellswithoneofthreepSinRep19-basedvectorRNAsex-pressingJEVstructuralproteinsandsubsequently

Library] At: 01:50 4 January 2008 JEV-basedforeigngenedeliveryS-IYunetal

531

selectingwithpuromycin.TheselectedcellsstablyexpressedJEVstructuralproteinswithnodeleteri-ouseffectsonthehostcells(datanotshown).ThesePCLsweremoreefficientinproducingJEVVRPsthanweretheparentalBHK-21cells.Inallcases,approximately5-to10-foldhigherVRPtiterswereobtainedupontransfectionofthesePCLs,withoneofsixJEVrepliconRNAsexpressingtheegfp,luc,orlacZgenebasedoneitherpJEV/Rep/󰀄C+󰀄prM+󰀄EorpJEV/Rep/󰀄CtoE,whencomparedtothecotrans-packagingcapacityofJEVisalsousefulincasetwoormoreexpressionunitsneedtobeadded(Agapovetal,1998).Ontheotherhand,wehavefoundthatthereplicationkineticsandfinalvirustitersincellcultureproportionallydecreasedastheinsertedgenelengthincreased.Ourdataindicatethatsizeisanon-specificfactoraffectingtheabilityoftheRNAtoserveasatemplateforreplication,withsmallerRNAsgen-erallybeingbettertemplates.Thesimplestexplana-tionforthiseffectisthatasthelengthoftheRNAytfectionoftheparentalBHK-21cellswithtwovectorisrRNAs(Figure7c).Inordertoverifythattheviruswasevtrulypropagationdeficient,weattemptedtoamplifyinUanyverylowamountsofpropagation-competent rvirusparticlesthatmighthavebeenpresent.Wedidetsnotdetectanypropagation-competentvirusparti-aMclesinourpackagingsystemafterthreepassagescoftheundilutedsupernatantcontaining3×105M[IUVRPsonna¨ıveBHK-21cells(datanotshown). :yThus,wehavesuccessfullydemonstratedthattrans-B complementation-basedpackagingsystemsforJEVderepliconvectorscanproducesingle-roundinfectious,dapropagation-deficientJEVVRPs.

olnwoDDiscussion

HerewereportthedevelopmentofaJEV-basedvec-torsystemforforeigngeneexpressionthatutilizesafull-lengthinfectiousJEVcDNA.Wehaveestab-lishedandfullycharacterizedthreestrategiesfortheexpressionofforeigngenesusingtheRNAreplica-tionmachineryofJEV.ThefirststrategymadeuseofrecombinantinfectiousvectorRNAs/virusesencod-ingforeigngenesofvarioussizes,preferentiallyatthevariableregionofJEV3′NTR,andshowedanin-versecorrelationbetweengenomelengthandgeneexpression.Thesecondapproachinvolvedavarietyofreplication-competent,propagation-deficientviralrepliconvectorRNAsshowingaspectrumofRNAreplicationefficiencies.Thethirdproceduremadeuseofstablepackagingcelllinesfortheproductionofsingle-roundinfectious,propagation-deficientJEVVRPs.ThesedatastronglysuggestthatourJEV-basedvectorsystemsrepresentattractivepotentialcandi-datesforforeigngeneexpressionandantiviralcom-poundscreeninginawidevarietyofcellsinvitro,andpossiblyinvivoforimmunizationapplications.WhenwedeterminedthemaximumextrasequencelengththatJEVcouldaccommodate,wefoundtooursurprisethatJEVwascapableofencapsidatingrecombinantJEVgenomicRNAsencodingheterolo-gousreportergenesaslargeas≈15kbintoinfectiousviralparticles.BecauseJEVrepliconRNAderivedfrompJEV/Rep/󰀄CtoEis≈9kbinsize,weestimatethataforeigngeneofatleast6kbcanbepackagedintoJEVVRPs.Thisresultisofparticularsignificancefortheexpressionoflengthygenes,suchasthecysticfi-brosistransmembraneconductanceregulator,whosecodingsequenceis≈4.5kb(Flotteetal,1993).Alarge

templateincreases,thenumberofminus-andplus-strandelongationcyclesalsoincreases;however,wecannotruleoutthepossibilitythatlargerRNAsrepli-catelessefficientlybecauseofdifferencesinGCcon-tentorintheirsecondaryand/ortertiarystructures.WehavealsofoundthattworecombinantJEVsharvestedfromBHK-21cellstransfectedwithpJEV/FL/3egfp-orpJEV/FL/3lacZ-derivedRNAarecapableoftransducingawidevarietyofcelltypes,includingSH-SY5Y(humanneuroblastoma),HeLa(humancervixadenocarcinoma),MOLT-4(humanlymphoblasticleukemia),Vero(monkeykidney),Neuro-2a(mouseneuroblastoma),MDCK(dogkid-ney),CRFK(catkidney),BHK-21(hamsterkidney),C6/36(mosquitolarva),andprimaryratneurons(datanotshown).Itisimportanttonote,however,thatforeigngenesinsertedatthebeginningoftheJEV3′NTRappearedtobeunstable,inthatthelongerforeigngenesmorerapidlylosttheiractivityduringserialpassages.WhenweinfectedBHK-21cellsatanmultiplicityofinfection(MOI)of1withtwore-combinantJEVsexpressingtheegfporlacZgene,wefoundthatthepercentageofinfectedcellsex-pressingbothJEVproteinsandthecorrespondingre-portergenedecreasedwitheachpassage,with≈50%(pJEV/FL/3egfp)and≈30%(pJEV/FL/3lacZ)ofthecellsexpressingbothproteinsatpassages3and2,respectively(datanotshown).Inlightofreportscon-cerningotherflavivirusvectorsystems(Fayzulinetal,2006;Gehrkeetal,2005;Piersonetal,2005),thisfindingwasnotunexpected,becausetheincreaseingenomesizewasassociatedwithadecreasedrateofviralreplicationandviceversa,providingtheoppor-tunityfordeletionofalloraportionoftheinsertedforeigngenethatisunnecessaryforviralreplication.Thus,althoughtheapplicationofsuchJEV-basedvec-torsystemsappearstobelimitedinpartbythelong-terminstabilityoftheviralgenomewhenitcontainsaforeigngeneofalargersize,thegenomeshouldstillbestableenoughtoproducerecombinantJEVscarry-ingsuchaforeigngeneduringthefirst3to4daysofRNAtransfection.

Vectorsbasedonself-replicatingrepliconsofsev-eralflaviviruseshaverecentlybeenconstructedandcharacterizedforpotentialapplicationtogeneex-pressioninmammaliancellsandthedevelopmentofnovelantiviraltherapeuticsandvaccines(Gehrkeetal,2005;Jonesetal,2005;KhromykhandWest-away,1997;Molenkampetal,2003;Pangetal,2001;Shietal,2002;VarnavskiandKhromykh,

Library] At: 01:50 4 January 2008 532

JEV-basedforeigngenedelivery

S-IYunetal

1999).Arelativelysmallgenomesizeandsim-pleprocedureallowforrapidgenerationofrecom-binants,andcontinuoussynthesisofdsRNAin-termediatesduringreplicationyieldsanenhancedimmuneresponse.Inthepresentstudy,wehavesystematicallymanipulatedthegenomeofJEVforthefirsttimetogenerateapanelofnineviralrepliconsbyintroducingonetothreeinternalin-framedeletionsintheectodomainsofstructuralpro-teins.Ofthese,allexceptpJEV/Rep/3luc/󰀄CCwereRNAproductionbypJEV/Rep/3luc/󰀄C+󰀄prM+󰀄EwiththatofpJEV/Rep/3luc/󰀄CtoE,whichlackstwopairsoftransmembranedomainsintheC-terminalportionsoftheprMandEproteinsremaininginpJEV/Rep/3luc/󰀄C+󰀄prM+󰀄E.RNAproductionbypJEV/Rep/3luc/󰀄CtoEwas≈6.5-foldhigherthanthatofpJEV/Rep/3luc/󰀄C+󰀄prM+󰀄Eat48hpost-transfection.Alternatively,itisconceivablethattheunnaturalrepeatingoftransmembranedomainsleadstoincorrectorinefficienttranslocationoftheNS1ytreplication-competent.ItisimportanttonotethatisrpJEV/Rep/3luc/󰀄CCisidenticaltopJEV/Rep/3luc/ev󰀄Cexceptforanadditional5′deletion(nt132toinU201)inthecodingregionoftheCprotein;therefore, rthisregionisapparentlyessentialforRNAreplica-etstion.ThisfindingisconsistentwiththeproposedaMcyclizationsequencemotifinthe5′regionoftheCcgene,whichisknowntoberequiredforreplicationM[inothermosquito-borneflaviviruses,suchasDENV, :yYFV,KUNV,andWNV(Alvarezetal,2005;Breden-B beeketal,2003;Corveretal,2003;Hahnetal,1987;deKhromykhetal,2001;Loetal,2003;Youetal,2001).daFurthermore,wehavealsoengineeredavarietyofolnJEVrepliconvectorRNAsthatarepackagedwhenwthestructuralproteinsaresuppliedintrans,usingaoDSindbisvirus-basedexpressionsystem,aspreviouslyreportedforotherflavivirusreplicon-derivedvectors(Fayzulinetal,2006;Gehrkeetal,2003;Harveyetal,2004;Khromykhetal,1998;Masonetal,2006;Scholleetal,2004).

Theeightself-replicatingluc-expressingJEVrepli-consconstructedheredisplayedawidespectrumofRNAreplicationandproteinexpressionandvari-ouslevelsofcytopathogenicity.Specifically,thelev-elsofRNAproductionandviralproteinaccumu-lationweresynergisticallyreducedbyaninternalin-framedeletionintroduced,eitherindividuallyorincombination,intotheectodomainsoftheCandprMproteinsofeitherpJEV/Rep/3luc/󰀄EorparentalpJEV/FL/3luc.Accordingtoourcurrentunderstand-ing,theendoplasmicreticulum(ER)istheprinci-palsiteatwhichJEVproteinsynthesis,virusassem-bly,andfinalmaturationtakeplace(LindenbachandRice,2001).JEVinfectioncausesextensiveprolifer-ationofthesecretoryapparatus,includingroughERandGolgicomplexes,inBHK-21cells(Wangetal,1997).TheERisextremelysensitivetochangesinhomeostasis,andinresponsetoavarietyofstim-uli,certainsignalsaretransducedfromtheERtoboththecytoplasmandnucleus,resultinginadap-tationforsurvivalorinductionofapoptosis(Kauf-man,1999;Pahl,1999).RecentstudieshaveshownthatJEVinfectioninducesERstress,triggeringtheunfoldedproteinresponse,whichinturninitiatesauniquesignalingcascadefromtheERtothenucleus(Suetal,2002).Accordingly,wehypothesizethatthetransmembranedomainsofJEVstructuralpro-teinsexpressedfromtheC-terminusintheERsup-pressthereplicationofeachviralrepliconRNA.Thishypothesiswassupportedbydirectcomparisonof

proteintotheroughER,therebypotentiallyinterfer-ingwiththebiogenesisoftheremainderportionofthepolyproteinandresultingintheinefficientRNAreplication.

Insummary,theJEV-basedvectorsystemdescribedinthisarticlerepresentsapromisingsystemforde-liveringforeigngenestocells,notonlyinvitrofortheexpressionofforeigngenesandscreeningofantivi-ralcompoundsbutpotentiallyalsoinvivoforthede-velopmentofgeneticvaccines.ThelargepackagingcapacityofJEVshouldfacilitatethedesignofvectorsforexpressinglargergenesandtreatmentofassoci-ateddiseases.Also,theunexpectedinfluenceofthetransmembranedomainswithintheviralstructuralproteinsonRNAreplicationmayprovidenovelinfor-mationregardingcross-talkthatconnectsthebiogen-esisoffunctionalstructuralproteinsofflaviviruseswithintheERandthelevelsofRNAproductionandgeneexpressioninthecytoplasmofinfectedcells.

Materialsandmethods

Cells

BHK-21cellsweremaintainedinalphaminimales-sentialmediumsupplementedwith10%fetalbovineserum(FBS),2mML-glutamine,vitamins,andan-tibiotics(Yunetal,2003).ThestableJEVrepliconpackagingcelllinesderivedfromBHK-21cellsweremaintainedinthepresenceof1µg/mlpuromycin.Constructionofplasmids

Allplasmidswereconstructedusingstandardmolec-ularbiologyprotocols,andtheregionsamplifiedbyPCRwereverifiedbysequencing.TherecombinantJEV-derivedvectorsusedinthisstudywerecon-structedbasedonpBACSP6/JVFLx/XbaI(Yunetal,2003),designated“pJEV/FL”inthisreport.Thenu-cleotidepositionreferstothecompletenucleotidese-quenceofJEVCNU/LP2(GenBankaccessionnumberAY585243).Onlythesalientfeaturesoftheseplas-midsaredescribedhere.Detailsofthecloningstrate-giesareprovidedinSupplementalMaterials,andcomputer-readablesequencefilesarealsoavailableuponrequest.

Recombinantfull-lengthJEVvectors:pJEV/FL/3-egfpwasconstructedbyinsertingtheEMCVIRES-drivenEGFPexpressioncassetteimmediatelydownstreamofthefirst41nucleotidesofJEV3′NTR,

Library] At: 01:50 4 January 2008 JEV-basedforeigngenedeliveryS-IYunetal

533

followedbytheentireJEV3′NTR.pJEV/FL/3luccontainedthelucgeneinlieuoftheegfpgeneofpJEV/FL/3egfp.pJEV/FL/3lucREP−wasidenticaltopJEV/FL/3luc,exceptthatan83-nucleotidedele-tion(nt5581to5663)wasintroducedintothemiddleoftheNS3gene,resultinginprematureterminationofviraltranslationatnt5596(Yunetal,2003).pJEV/FL/3lacZcontainedthelacZgeneencodingthebacterialβ-galactosidaseenzymeinexchangeforthecodingsequenceofegfpofnomicRNApromoter.pSinRep19/JEVC-E-BglIIcon-tainedthecodingsequenceofJEVCthroughEplusthe58N-terminalresiduesofNS1,whereaspSin-Rep19/JEVC-NS1includedthecompletecodingse-quenceofJEVCthroughNS1.

RNAtranscriptionandtransfection

PurifiedpJEV/FLanditsderivativeswerelinearizedbydigestionwithXbaI,followedbytreatmentwithmungbeannuclease.PurifiedpSinRep19anditsytpJEV/FL/3egfp.

isrevPropagation-deficientJEVrepliconvectors:ApanelinUofnineJEVrepliconvectorsexpressingthelucgene rwasconstructedtofacilitatethemonitoringofrepli-etscation.Thesevectors,basedonpJEV/FL/3luc,wereaMproducedbyintroducingonetothreeinternalin-cframedeletionsinthecodingsequencesofparticularM[JEVstructuralproteins.Alldeletionswereidentified :ybyanovelXhoIsitethatresultedintheinsertionB ofLeuandGlu.Thefirstsetoffourviralreplicondevectors(pJEV/Rep/3luc/󰀄CC,pJEV/Rep/3luc/󰀄C,dapJEV/Rep/3luc/󰀄prM,andpJEV/Rep/3luc/󰀄E)con-olntaineda273-nucleotidedeletion(nt132to404)wintheCgene,a204-nucleotidedeletion(nt201oDto404)intheCgene,a282-nucleotidedele-tion(nt531to812)intheprMgene,anda1170-nucleotidedeletion(nt1032to2201)intheEgene,respectively.Thesecondsetofthreeviralrepliconvectors(pJEV/Rep/3luc/󰀄C+󰀄prM,pJEV/Rep/3luc/󰀄C+󰀄E,andpJEV/Rep/3luc/󰀄prM+󰀄E)containedapairofinternalin-framedeletionsinthecodingregionsoftwoJEVstructuralproteinsandaredesignatedaccordingly.Thethirdsetoftwovi-ralrepliconvectors(pJEV/Rep/3luc/󰀄C+󰀄prM+󰀄EandpJEV/Rep/3luc/󰀄CtoE)lackedallthreeJEVstructuralproteins.pJEV/Rep/3luc/󰀄C+󰀄prM+󰀄Econtainedallthreeinternalin-framedeletions,namelya204-nucleotidedeletion(nt201to404)intheCgene,a282-nucleotidedeletion(nt531to812)intheprMgene,anda1170-nucleotidedeletion(nt1032to2201)intheEgene.pJEV/Rep/3luc/󰀄CtoEcontaineda2,001-nucleotidedeletion(nt201to2201)intheviralgenome,excludingthe35N-terminaland24C-terminalaminoacidsoftheCpro-teinandfollowedimmediatelybytheN-terminusoftheNS1proteinandtherestoftheviralgenome.Wealsogeneratedaseparatesetoffourviralrepliconvectors(pJEV/Rep/3egfp/󰀄C+󰀄prM+󰀄E,pJEV/Rep/3egfp/󰀄CtoE,pJEV/Rep/3lacZ/󰀄C+󰀄prM+󰀄E,andpJEV/Rep/3lacZ/󰀄CtoE)designedtoexpresseitheregfporlacZ,byreplacingthelucgeneofpJEV/Rep/3luc/󰀄C+󰀄prM+󰀄EorpJEV/Rep/3luc/󰀄CtoEwiththecorrespondingreportergene.

JEVstructuralproteinexpressionvectors:pSin-Rep19(Agapovetal,1998),aSindbisvirus–basedheterologousgeneexpressionvector,wasengineeredtoexpressthreeJEVstructuralproteins:pSin-Rep19/JEVC-EcontainedtheentirecodingsequenceofJEVCthroughEunderthecontrolofitsownsubge-

derivativeswerelinearizedbydigestionwithXhoI.Linearizedplasmidswereusedforinvitrotranscrip-tionreactionsemployingSP6RNApolymerase,asdescribedpreviously(Yunetal,2003).Aftertran-scription,reactionmixtureswerefurtherincubatedwith10UDNaseIfor30minandextractedwithphenol-chloroform-isoamylalcohol.RNAyieldwasquantifiedonthebasisof[3H]UTPincorporation,asmeasuredbyRNAabsorptiontoDE-81filterpaper(Whatman,Maidstone,UK).RNAintegritywasas-sessedbyagarosegelelectrophoresis.RNA(2µg)wastransfectedintocellsbyelectroporation,asdescribedpreviously(Yunetal,2003).

Real-timequantitativeRT–PCR

TotalRNAwasextractedfromduplicatewellswithTRIzolreagent(Invitrogen,Carlsbad,CA).Specifi-cally,50ngoftotalcellularRNAwasusedforreversetranscriptionusingSuperscriptIIRT(Gibco-BRLLifeTechnologies,Gaithersburg,MD)withprimersspe-cificfortheJEVNS3region,aswellasBHK-21β-actinRNAtonormalizetotalRNAlevels.JEVandBHK-21β-actincDNAsweregeneratedbyreversetranscrip-tionat45◦Cfor30min,followed◦byinactivationofreversetranscriptaseat95Cfor10min.JEV-specificandBHK-21β-actin–specificcDNAswereamplifiedwiththeiQSupermixQuantitativePCRSystem(Bio-RadLaboratories,Hercules,CA)anddetectedwiththeiCycleriQMulticolorReal-TimePCRDetectionSystem(Bio-RadLaboratories).Afraction(1/10th)ofthereversetranscriptionreactionwasusedfor50cyclesofamplificationat95◦Cfor15sand60◦Cfor1min.JEVforwardandreverseprimerswere5′-ATCCAACTCAACCGCAAGTCand5′-TCTAAGATG-GTGGGTTTCACG,respectively.Theprobesequence(nt5837to5856)was5′-6FAMCATCTCTGAAATGG-GGGCTA-BHQ1(IntegratedDNATechnologies,Cor-alville,IA).TheforwardandreverseprimersforBHK-21β-actinwere5′-ACTGGCATTGTGATGGA-CTCand5′-CATGAGGTAGTCTGTCAGGTC,respec-tively.Theprobesequencewas5′-HEX-CCAGCCAG-GTCCAGACGCAGG-BHQ2(IntegratedDNATech-nologiesInc.).The2−󰀄󰀄CTmethodwasusedtoanalyzerelativechangesinJEVRNAlevelsfromreal-timequantitativeRT-PCRexperiments(Wineretal,1999).

Immunoblotting

Theexperimentalproceduresusedaredescribedindetailelsewhere(Yunetal,2003).One-twentieth

Library] At: 01:50 4 January 2008 534

JEV-basedforeigngenedelivery

S-IYunetal

(4×105cells)oftheelectroporatedcellsdescribedabovewasplatedona6-wellplate.Atthein-dicatedtimepointsaftertransfection,cellswerelysedwith200µlof1×sampleloadingbuffer(80mMTri-HCl[pH6.8],2.0%sodiumdodecylsul-fate[SDS],10%glycerol,0.1Mdithiothreitol[DTT],0.2%bromophenolblue).One-tenthofthelysatewasresolvedbyelectrophoresisonSDS–12%poly-acrylamidegelsandelectroblottedontomethanol-activatedpolyvinylidenedifluoridemembranes.JEVFlowcytometryanalysis:Cellsweretrypsinized,washedoncewithPBS,andresuspendedin0.37%(v/v)formaldehydeinPBS,followedbyanalysisofegfpexpressionusingaFACSCaliburflowcytome-ter(BDBiosciences,SanJose,CA).Deadcellswereexcludedbyappropriategatingforforwardandsidelight-scattering,and1×104viablecellswerecountedpersample.

Luciferaseassay:Luciferaseactivity,withluciferinytproteinswereprobedwithamousehyperimmuneisrantiserum(1:1000dilution)specificforJEV,ob-evtainedfromtheAmericanTypeCultureCollectioninU(VR-1259AF).JEVNS1proteinswerestainedwith rarabbitantiserum(1:1000dilution)raisedagainstetstheglutathioneS-transferase-fusedN-terminal166aMaminoacidsofJEVNS1protein(nt2478to2975).cGAPDHproteinsweredetectedwitharabbitanti-M[serum(1:5000dilution)fromtheLabFrontier,Seoul, :yKorea.TheimmunoreactiveproteinswerevisualizedB bythebindingofalkalinephosphatase-conjugateddegoatanti-mouseoranti-rabbitIgGs(1:1000dilu-dation;JacksonImmunoResearchLabs,WestGrove,olnPA)andsubsequentincubationwiththesubstratesw5-bromo-4-chloro-3-indolylphosphateandnitroblueoDtetrazolium.

Expressionofheterologousreportergenesofvarioussizes

Immunofluorescencemicroscopy:Foregfpexpres-sion,cellswereseededina4-wellchamberslidefor36to48h.Afterincubation,theywerefixedinphosphate-bufferedsaline(PBS)containing0.37%(v/v)formaldehydeandmountedin0.2mlof80%glycerol.ImagesweregeneratedonaZeissAx-ioskopconfocalmicroscopeequippedwithafluores-ceinfilterusingBio-RadMRC1024andLaserSharpsoftware.

References

AgapovEV,FrolovI,LinderbachBD,PragaiBM,SchlesingerS,RiceCM(1998).NoncytopathicSindbisvirusRNArepliconsforheterologousgeneexpression.AlvarezProcNatlDE,AcadLodeiroSciUMF,SALuduena95:129–12994.

SJ,PietrasantaLI,GamarnikAV(2005).Long-rangeRNA-RNAinteractionscircularizethedenguevirusgenome.JVirol79:6631–63.

BonaldoMC,GarrattRC,CaufourPS,FreireMS,RodriguesMM,NussenzweigRS,GallerR(2002).Surfaceexpres-sionofanimmunodominantmalariaproteinBcellepi-topebyyellowfevervirus.JMolBiol315:873–885.BonaldoMC,GarrattRC,MarchevskyRS,CoutinhoES,Ja-borAV,AlmeidaLF,YamamuraAM,DuarteAS,OliveiraPJ,LizeuJO,CamachoLA,FreireMS,GallerR(2005).Attenuationofrecombinantyellowfever17Dvirusesex-pressingforeignproteinepitopesatthesurface.JVirol79:8602–8613.

assubstrate,wasmeasuredincelllysatesbyusingacommercialassayaccordingtothemanufacturer’srecommendations(Promega,Madison,WI).Eachex-perimentwasperformedintriplicate,andthemeanvaluesarepresented.

β-Galactosidaseassay:CellswerewashedoncewithPBS,fixedwith0.05%(v/v)glutaraldehydeinPBSfor15minatroomtemperature,andcare-fullywashedthreetimeswithPBS.Theywereassessedforβ-galactosidaseactivitybyincuba-tioninstainingsolution(5mMpotassiumfer-ricyanide,5mMpotassiumferrocyanide,2mMMgCl2inPBS)and5-bromo-4-chloro-3-indolyl-β-galactopyranoside(Sigma-Aldrich,St.Louis,MO)at37◦C.

GenerationofstablepackagingcelllinesforJEVrepliconRNAsNa¨ıveBHK-21cellsweretransfectedwithpSinRep19-basedJEVstructuralproteinexpres-sionvectorRNAs,asspecifiedinthetext.Aftertransfection,cellswereseededfor≈24h,andthemediumwasreplacedwithfreshcompletemediumcontaining10µg/mlpuromycin.There-after,cellsweremaintainedinthepresenceof1µg/mlpuromycinandeitherpassagedorfrozen,asweretheparentalBHK-21cells.

BredenbeekPJ,KooiEA,LindenbachB,HuijkmanN,RiceCM,SpaanWJ(2003).Astablefull-lengthyellowfeverviruscDNAcloneandtheroleofconservedRNAel-ementsinflavivirusreplication.JGenVirol84:1261–1268.

BredenbeekPJ,MolenkampR,SpaanWJ,DeubelV,Mari-anneauP,SalvatoMS,MoshkoffD,ZapataJ,TikhonovI,PattersonJ,CarrionR,TicerA,BraskyK,Lukashe-vichIS(2006).Arecombinantyellowfever17DvaccineexpressingLassavirusglycoproteins.Virology345:299–304.

CorverJ,LenchesE,SmithK,RobisonRA,SandoT,StraussEG,StraussJH(2003).Finemappingofacis-actingse-quenceelementinyellowfevervirusRNAthatisre-quiredforRNAreplicationandcyclization.JVirol77:2265–2270.

FayzulinR,ScholleF,PetrakovaO,FrolovI,MasonPW(2006).Evaluationofreplicativecapacityandgenetic

Library] At: 01:50 4 January 2008 JEV-basedforeigngenedeliveryS-IYunetal

535

stabilityofWestNilevirusrepliconsusinghighlyeffi-cientpackagingcelllines.Virology351:196–209.

FlotteTR,AfioneSA,SolowR,DrummML,MarkakisD,GugginoWB,ZeitlinPL,CarterBJ(1993).Expressionofthecysticfibrosistransmembraneconductanceregulatorfromanoveladeno-associatedviruspromoter.JBiolChem268:3781–3790.

GehrkeR,EckerM,AberleSW,AllisonSL,HeinzFX,MandlCW(2003).Incorporationoftick-borneen-cephalitisvirusrepliconsintovirus-likeparticlesbyapackagingcellline.JVirol77:24–33.

MarkoffL(2003).5′-and3′-noncodingregionsinflavivirusRNA.AdvVirusRes59:177–228.

MasonPW,ShustovAV,FrolovI(2006).Productionandcharacterizationofvaccinesbasedonflavivirusesde-fectiveinreplication.Virology351:432–443.

MolenkampR,KooiEA,LucassenMA,GreveS,ThijssenJC,SpaanWJ,BredenbeekPJ(2003).YellowfevervirusrepliconsasanexpressionsystemforhepatitisCvirusstructuralproteins.JVirol77:14–18.

MonathTP(2003).Yellowfevervaccine,4thed.Philadel-phia:WBSaunders.

yGehrkeR,HeinzFX,DavisNL,MandlCW(2005).Heterolo-tisgousgeneexpressionbyinfectiousandrepliconvectorsrevderivedfromtick-borneencephalitisvirusanddirectincomparisonofthisflavivirussystemwithanalphavirusUreplicon.JGenVirol86:1045–1053.

reHahnCS,HahnYS,RiceCM,LeeE,DalgarnoL,StraussEG,tsStraussJH(1987).Conservedelementsinthe3′untrans-aMlatedregionofflavivirusRNAsandpotentialcyclizationcsequences.JMolBiol198:33–41.

M[HarveyTJ,LiuWJ,WangXJ,LinedaleR,JacobsM,David- :ysonA,LeTT,AnrakuI,SuhrbierA,ShiPY,KhromykhB AA(2004).Tetracycline-induciblepackagingcelllinedeforproductionofflavivirusrepliconparticles.JVirol78:da531–538.

oHennessyS,LiuZ,TsaiTF,StromBL,WanCM,LiuHL,WulnwTX,YuHJ,LiuQM,KarabatsosN,BilkerWB,HalsteadoSB(1996).Effectivenessoflive-attenuatedJapaneseen-Dcephalitisvaccine(SA14-14-2):acasecontrolstudy.JonesLancetCT,Patkar347:1583–1586.

CG,KuhnRJ(2005).Constructionandap-plicationsofyellowfevervirusreplicons.Virology331:247–259.

KaufmanRJ(1999).Stresssignalingfromthelumenoftheendoplasmicreticulum:coordinationofgenetranscrip-tionalandtranslationalcontrols.GenesDev13:1211–1233.

KhromykhAA,MekaH,GuyattKJ,WestawayEG(2001).EssentialroleofcyclizationsequencesinflavivirusRNAreplication.JVirol75:6719–6728.

KhromykhAA,VarnavskiAN,WestawayEG(1998).Encap-sidationoftheflavivirusKunjinrepliconRNAbyusingacomplementationsystemprovidingKunjinvirusstruc-turalproteinsintrans.JVirol72:5967–5977.

KhromykhAA,WestawayEG(1997).Subgenomicrepli-consoftheflavivirusKunjin:constructionandapplica-tions.JVirol71:1497–1505.

LaiCJ,MonathTP(2003).Chimericflaviviruses:novelvac-cinesagainstdenguefever,tick-borneencephalitis,andJapaneseencephalitis.AdvVirusRes61:469–509.

LindenbachBD,RiceCM(2001).Flaviviridae:Thevirusesandtheirreplication.In:Fieldsvirology,4thed.KnipeDM,HowleyPM,GriffinDE,LambRA,MartinMA,RoizmanB,StrausSE(eds).Philadelphia:LippincottWilliams&WilkinsPublishers,pp991–1041.

LoMK,TilgnerM,BernardKA,ShiPY(2003).Func-tionalanalysisofmosquito-borne′flavivirusconservedsequenceelementswithin3untranslatedregionofWestNilevirusbyuseofareportingrepliconthatdifferen-tiatesbetweenviraltranslationandRNAreplication.JVirol77:10004–10014.

PahlHL(1999).Signaltransductionfromtheendoplas-micreticulumtothecellnucleus.PhysiolRev79:683–701.

PangX,ZhangM,DaytonAI(2001).DevelopmentofdenguevirusrepliconsexpressingHIV-1gp120andotherheterologousgenes:apotentialfuturetoolfordualvaccinationagainstdenguevirusandHIV.BMCMicro-Piersonbiol1:TC,28–36.

DiamondMS,AhmedAA,ValentineLE,DavisCW,SamuelMA,HannaSL,PufferBA,DomsRW(2005).AninfectiousWestNilevirusthatexpressesaGFPre-portergene.Virology334:28–40.

ScholleF,GirardYA,ZhaoQ,HiggsS,MasonPW(2004).transproperties-PackagedinvitroWestandNileinvirus-likeinfectedparticles:mosquitoinfectiousvectors.JShiVirolPY,Tilgner78:11605–11614.

M,LoMK(2002).Constructionandcharac-terizationofsubgenomicrepliconsofNewYorkstrainofWestNilevirus.Virology296:219–233.

SolomonT(2003).RecentadvancesinJapaneseencephali-tis.JNeuroVirol9:274–283.

SuHL,LiaoCL,LinYL(2002).Japaneseencephalitisvirusinfectioninitiatesendoplasmicreticulumstressandanunfoldedproteinresponse.JVirol76:4162–4171.

VarnavskiAN,KhromykhAA(1999).Noncytopathicfla-vivirusrepliconRNA-basedsystemforexpressionanddeliveryofheterologousgenes.Virology255:366–375.

WangJJ,LiaoCL,ChiouYW,ChiouCT,HuangYL,ChenLK(1997).UltrastructureandlocalizationofEproteinsinculturedneuroncellsinfectedwithJapaneseencephali-tisvirus.Virology238:30–39.

WinerJ,JungCK,ShackelI,WilliamsPM(1999).De-velopmentandvalidationofreal-timequantitativere-versetranscriptase-polymerasechainreactionformoni-toringgeneexpressionincardiacmyocytesinvitro.AnalXinBiochemYY,Ming270:ZG,41–49.

PengGY,JianA,MinLH(1988).Safetyofalive-attenuatedJapaneseencephalitisvirusvaccine(SA14-14-2)forchildren.AmJTropMedHyg39:214–217.

YouS,FalgoutB,MarkoffL,PadmanabhanR(2001).InvitroRNAsynthesisfromexogenousdengueviralRNAtemplatesrequireslongrangeinteractionsbetween5′-and3′-terminalregionsthatinfluenceRNAstructure.JYunBiolSI,ChemKimSY,276:Rice15581–15591.

CM,LeeYM(2003).DevelopmentandapplicationofareversegeneticssystemforJapaneseencephalitisvirus.JVirol77:50–65.