ProcNatiAcadSciUSAVol87pp29702974April1990MedicalSciencesAtransactiv - PDF document

1 Proc.Nati.Acad.Sci.USAVol.87,pp2970-2974
Proc.Nati.Acad.Sci.USAVol.87,pp2970-2974,April1990MedicalSciencesAtrans-activatorfunctionisgeneratedbyintegrationofhepatitisBviruspreS/SsequencesinhumanhepatocellularcarcinomaDNA(trans-activation/truncation/pSV2CATreporterplasmid)WOLFGANGH.CASELMANN*tt,MARKUSMEYER*,ALEXANDERS.KEKULO*,ULRICHLAUER*,PETERH.HOFSCHNEIDER*,ANDRAJENKOSHY*t*Max-Planck-InstitutfurBiochemie,DepartmentofVirusResearch,D-8033Martinsried,FederalRepublicofGermany;andtDepartmentofMedicineII,KlinikumGrosshadern,UniversityofMunich,D-8000Munich70,FederalRepublicofGermanyCommunicatedbyCharlesWeissmann,December28,1989(receivedforreviewOctober30,1989)ABSTRACTTheXgeneofwild-typehepatitisBvirusorintegratedDNAhasrecentlybeenshowntostimulatetran-scriptionofavarietyofenhancersandpromoters.Tofurtherdelineatetheviralsequencesresponsiblefortrans-activationinhepatomas,weclonedthesinglehepatitisBvirusinsertfromhumanhepatocelluiarcarcinomaDNAMl.TheplasmidpMlcontains2004basepairsofhepatitis'BvirusDNAsubtypeadr,including'truncatedpreS/Ssequencesandtheenhancer'ele-ment.'TheXpromoterand422nucleotidesoftheXcodingregionarepresent.TheentirepreC/Cgeneisdeleted.IntransientcotransfectionassaysusingChanglivercefls'(CCL13),pMlDNAexerts'a6-to10-foldtrans-activatingeffectontheexpressionofthepSV2CATreporterplasmid.Thetrans-activationoccursbystimulationoftranscriptionandisdepen-denton'thesimianvirus40enhancerinthereporterplasmid.Deletionanalysis'ofpMlsubclonesrevealsthatthetrans-activatoris'encodedbypreS/SandnotbyXsequences.AframeshiftmutationwithinthepreS2openreadingframeshowsthatthisportionisindispensableforthetrans-activatingfunction.InitiationoftranscriptionhasbeenmappedtotheSipromoter.Acomparabletrans-activatingeffectisalsoob-servedwithclonedwild-typehepatitisBvirussequencessim-ilarlytruncated.Theseresultsshowthatatranscriptionaltrans-activatorfunctionnotpresentintheintactgeneisgeneratedby3'truncationofintegratedhepatitisBvirusDNApreS/Ssequences.Strongepidemiologicalevidencecloselylinksthedevelop-mentofhepatocellularcarcinoma(HCC)withchronichep-atitisBvirus(HBV)infection(1).ClonallyintegratedHBVDNAsequencesarefoundinHCCtissueofmorethan90oofhepatitisBsurfaceantigen(HBsAg)-seropositivepatients(2).TheintegrationofHBVDNAisthusconsideredtobeapathogeneticfactorfor'tumordevelopment.InhumanHCCscis-activationofoncogenesbyHBVpro-moterorenhancerinsertioncannotberegardedasageneralmechanismoftransformation,althoughacaseisreportedwhereacellulargenerelatedtoaknownoncogenemighthavebeenactivatedbyintegratedHBVDNA(3).Ontheotherhand,HBVlikemanyotherhumanDNAviruses(4)bearstrans-activationalpotential.TheHBVXopenreadingframe(ORF)'encodesatranscriptionaltrans-activatorthatstimulatestranscriptionfromitsownandmanyheterologous'promotersintransienttransfectionassays(5-7).Thetrans-activatingfunctionofatumor-derivedHBVsubclonewasconserved,evenwhenXsequenceshadbeentruncatedbyintegration(8).Toextendtheseobservationsandtodelineatetheviralse-quencesresponsible,weclonedasingleHBVDNAinsertfromhumanhepatomatissue.Hereweprovideevidenceforatranscriptionaltrans-activatorencodedwithinthepreS/Sre-gionofHBVDNAwhoseactivityisnewlygeneratedbydislocationfromitsdownstreamsequences.MATERIALSANDMETHODSTissueofthehepatomadesignatedMlwasobtainedbypartialhepatectomyfroma46-year-oldChinesemanwithahistologicallyconfirmedHBsAg-positiveHCC.DNAExtractionandSouthernBlotHybridization.TenmicrogramsofhighmolecularweightgenomicMlDNAwasdigestedwithrestrictionendonucleases,electrophoresedin0.8%agarosegels,andtransferredtonitrocellulosemem-branes(9).Hybridizationwascarriedoutin50o(vol/vol)formamide,5xSSC(lxSSC=0.15Msodiumchloride/0.015Msodiumcitrate,pH7),and1%sarcosylat420CbyusingDNAprobeslabeledwith32Pbyrandompriming(10).Filterswereautoradiographedfor4-24hrat-700C.GenomicLibraryConstruction.HindIII-digestedMlDNAwasligatedintotheHindIllarmsofAvectorCharon28.RecombinantbacteriophageswerepackagedandadsorbedtoEscherichiacoli490-A'cells.Thelibrarywasscreenedwith32P-labeledHBVDNA,andtheDNAfromaplaque-purifiedpositiveclonewasreclonedinthepUC19vectortocreateplasmidpMl.PlasmidConstructionandSequencing.AllplasmidswereclonedinthepUC19vectorbyusingstandardmethodsunlessotherwisestated.ThenucleotidesequencesofpMlanditssubclonesweredeterminedbydideoxysequencing(11).ThecomputersoftwarepackageoftheUniversityofWisconsinGeneticsComputerGroup(UWGCG)andthenucleotidesequencesoftheEuropeanMolecularBiologyLaboratory(EMBL)DataLibrary(release20.0)wereusedforcomparisonofsequencehomologies.TransientExpressionofPlasmidDNAs.Changcells(ATCCCCL13)originallyderivedfromnonmalignanthumanlivercells(12,13)wereculturedinDulbecco'smodifiedEagle'smediumsupplementedwith10%(vol/vol)fetalcalfserum.FivepicomolesoftherespectiveplasmidDNAwastrans-fectedbythecalciumphosphatetechnique(14).Cellswereharvested12-18hrlaterandlysedinguanidiniumisothi

2 o-cyanate.TotalcellularRNAwaspurifiedbyC
o-cyanate.TotalcellularRNAwaspurifiedbyCsCIgradientcentrifugation(15)andtreatedwithRNase-freeDNase.Poly(A)+RNAwasisolatedthrougholigo(dT)-cellulosecol-umns.Onemicrogramofpoly(A)+RNAwaselectrophoresedinagarosegelswith20mM4-morpholinepropanesulfonicacid(Mops),5mMsodiumacetate,and0.1%EDTA,blottedtonylonmembranes,andhybridizedasdescribedabove.Thepublicationcostsofthisarticleweredefrayedinpartbypagechargepayment.Thisarticlemustthereforebeherebymarked"advertisement"inaccordancewith18U.S.C.§1734solelytoindicatethisfact.2970Abbreviations:HBV,hepatitisBvirus;HCC,hepatocellularcarci-noma;CAT,chloramphenicolacetyltransferase;SV40,simianvirus40;ORF,openreadingframe;HBsAg,hepatitisBsurfaceantigen.tTowhomreprintrequestsshouldbeaddressedatthe*address. Proc.Natl.Acad.Sci.USA87(1990)2971AB123-9.5-6.7�4.8--4.5-2.3-7.5-4.42.5---2.4-1.4FIG.1.(A)SouthernblotanalysisofhumanHCCMIDNArevealedasingleHBVbandof4.8kbandnoepisomalHBVDNA.(B)Northernblotanalysisofpoly(A)+RNAisolatedfrompMl-transfectedCCL13cellsshowedaviral-cellularread-throughtran-scriptof2.5kb,whichhybridizedwithtotalHBV(lane1)andpreS/S-specificprobes(nucleotides2844-127,lane2).AnXgene-specificprobe(nucleotides1400-1984,lane3)failedtohybridize.Molecularsizemarkers(inkb)areprovidedontheright.InVitroTranscriptionandRNaseProtectionMapping.Theappropriatetemplateplasmidwasusedtosynthesize[32Ip]UTP-labeledriboprobescomplementarytothemRNAtran-scribedfromthetransfectedtestplasmidswithSP6polymer-ase.About1.2x106CCL13cellswerecotransfectedwith2,ugofpSV2CATanda10-foldmolarexcessoftestplasmid.ThirtymicrogramsoftotalcellularRNAwashybridizedto1x106cpmoftheriboprobefor12-15hrin50o(vol/vol)deionizedformamide,40mMPipes(pH6.6),and400mMNaClat420C.AftertreatmentwithRNaseA(20gg/ml)andRNaseT1(5000units/ml)for1hrat370C,theprotectedfragmentswereanalyzedon6%polyacrylamide/8Mureagels(16).Autoradiographywasperformedat-700C.ChloramphenicolAcetyltransferase(CAT)Assay.Onemi-crogramofthereporterplasmidpSV2CAT(17)wascotrans-fectedwitha10-foldmolarexcessoftestplasmidintoCCL13cells.Fortyto48hraftertransfection,thecellswerelysedbysonication.Fiftymicrogramsoftotalproteinwasincubatedat370Cfor90mininthepresenceof4mMacetylcoenzymeAand75nCi(1Ci=37GBq)of[14C]chloramphenicol.TLCofthereactionproductswasperformedasdescribed(17).Allexper-imentswererepeatedfourtosixtimeswithatleasttwodifferentpreparationsofDNA.AcetylationwasquantifiedbycuttingouttheappropriateportionsoftheTLCplateanddeterminingtheradioactivityinaliquidscintillationcounter.RESULTSStructuralOrganizationofClonepMl.Southernblothy-bridizationofHindIII-digestedgenomicDNAfromHCCtissueMlrevealedasingle4.8-kilobase(kb)fragmentthathybridizedtoHBVDNA(Fig.LA).AnMlgenomiclibrarywasconstructedandscreenedwith32P-labeledHBVDNA.DNAfromaplaque-purifiedpositiveclonewasclonedinthepUC19vector,yieldingplasmidpM1.ThenucleotidesequenceofpMlwasdeterminedbydideoxysequencing(11).TheinsertinclonepM1consistsof2004basepairs(bp)ofHBVDNAwithanoverallhomologyof97.9%6totheHBVsubtypeadr(18).TheintegratedHBVDNAcontainsatruncatedpreS/Sgene(nucleotides2821-423and1000bpI~~~~~IsU-Chv-DothetlcaaQ0905bpdeletion-nZ'4/ZYAl4...COCTOupolyAsiteNviral-cellularfusiontranscrictsinitiatingatthe:XpromoterCpromoterSipromoterS2promoter3.05kbsize2.70kbsize2.50kbsize2.10kbsizeFIG.2.StructuralorganizationofclonepM1.TheindicatednucleotidepositionsrefertotheHBVinsert.BoxesrepresentintegratedHBVDNA.The905-bpdeletioncoveringtheCgeneisdepicted.Thetriangleindicatesa1-bpdeletionthatshiftedtheXORF.Solidbarsrepresenttheflankingcellularregions.ThetranscriptionalstartsitesattheX,C,S1,andS2promoters;thesizesofrespectiveviral-cellularfusiontranscripts;andthelocationofacellularpoly(A)siteareshown.AphysicalmapoftheHBVgenomeisalsoshown.P,promoter;E,enhancer;DRI/I,directrepeatI/II;a,AUGstartcodon.4.8kbr7-Y-Y-,leII..-T-"I.-.II11IV-I.---~~~~~~~~~~~~~~~~~~~~~MedicalSciences:Caselmannetal.preSIS21coLOt-'n-4COI-L1L-'L;--I 2972MedicalSciences:Caselmannetal.717-832;nomenclatureofGalibert,ref.19)withtheS1(20)andS2(20,21)promoters.Theviralenhancerelement(22)andtheXpromoter(23)areconserved.Twelve3'nucleotides(1797-1808)oftheXORFaremissing.TherecentlyidentifiedORF5(24)isintact.DeletionoftheentirepreC/CgenefusedpreSsequencesdirectlytotheXORF.The5'viralintegra-tionsitemapstonucleotide717andthe3'viralendmapstonucleotide423,bothlocatedinthesingle-strandedgapregionwithintheSgene,whichisoneofthepreferredsitesforrecombination(ref.25;Fig.2).Computeranalysisofthesequenced5'and3'flankingcellularDNArevealednohomologywithanyknownhumangenes.TranscriptionalTrans-ActivatingFunctionofpreS/SSe-quences.IntransientcotransfectionassaysusingtheCCL13cellline,pM1DNAstimulatedtheexpressionofpSV2CATDNA6-to10-foldascomparedtothepUC19vectorDNAalone(Fig.3).ThereporterplasmidpSV2CAT(17)containstheCATgeneunderthecontrolofthe

3 simianvirus40(SV40)earlypromoterandenhan
simianvirus40(SV40)earlypromoterandenhancer,whereaspSV1CATislackingtheSV40enhancer(17).NoactivationoftheCATgeneexpressionwasdetectablewhenpSV1CATwasused.ThesedatasuggestthattheSV40enhancerelementisnecessaryfortheeffect.InitiallyassumingthattheactivationwasduetoXse-quences,weconstructedsubclonepM35(Fig.3)containingHBVDNAfromnucleotides948-17%(i.e.,eliminatingallbuttheXORFandORF5sequencesandtheirreg-ulatoryelements).pM35didnotstimulatetheCATgeneexpressionsignificantlyhigherthanthecontrols.Thismightbeduetoa1-bpdeletionatposition1725thatshiftsthereadingframeandresultsinatranslationalstopatnucleotide1758.TheplasmidspFrandpFI(Fig.3)coveringthe5'and3'cellularflankingsequences,respectively,exertednoac-tivatingfunctioneither(Fig.3).TotesttheremainingHBVsequencesofpM1fortrans-activationalproperties,sub-clonespM18(nucleotides717-1236)andpM27(nucleotides1372-1796/2703-247)wereconstructed(Fig.3).WhereasplasmidpM18showednostimulatoryeffectonCATgeneexpression,pM27,comprisingtheXgenewithoutits5'regulatoryelementsandthepreS/Sregion,exertedasimilaractivatingeffect(averageof6.3-foldstimulation)astheentirepM1clone(Fig.3).SubclonepM56,whichcontainsthepreS/Ssequences(nucleotides1764-1796and2703-217)andbothSpromotersstimulatedCATgeneexpression7.1-foldwithrespecttothepUC19control(Fig.3).AframeshiftmutantpM56fsXhoIconstructedbycuttingtheDNAattheXhoIsite(nucleotide127)withinthepreS2ORFlostthetrans-activatingfunction(Fig.3).ThesedatasuggestthatthestimulatoryeffectonCATgeneexpressionismediatedbymeansofatrans-actinggeneproductofpreS/SspecificityandthatthepreS2portionisindispensablefortrans-activation.Thetruncatedproteinwouldcontaintheknownmyristoylationsite(26)andtheN-terminalaminoacidsofthelargeSprotein(27),bothofwhichhavebeensuggestedtoinhibitthesecretionoftheSprotein.Ontheotherhand,thepresenceof14of15aminoacidsofthemembranetranslo-cationsignal(aminoacids8-22ofthemajorSprotein,ref.28)andthelossofthemembranestopsignal(aminoacids80-98ofmajorSprotein,ref.28)couldresultinsecretionofthetrans-activatorprotein.However,withstandardHBsAgRIAs(AusriaII,Abbott),nocross-reactiveantigensweredetectablesofarinsupernatantsofCCL13cellstransfectedwithpM1.Toinvestigatethemodeofthetrans-activation,CAT-specifictranscriptsweremeasuredbyRNaseprotectionanalysisaftercotransfectionofCCL13cellswithpSV2CATandvariouspreS/S-containingtestplasmids.TransfectionwithplasmidspM1,pM27,orpM56ledtoelevatedlevelsofNOccODNC)CONNNCO0C\EE-U--3\o-_I).AlIXpreSlS2S1).1(")(!-rI.:".;.140^.*...S_pOf?81.0P'3,1~-)JP.t)I.,p;t:f,fXh'-,wildtypeHBV:.4iFIG.3.CATassaysfortrans-activationbypM1,subclonesofpM1,andwild-typeHBVDNA.ThetoplinerepresentspM1.SolidlinesbelowthemapsymbolizepM1subclones.TheinvertedtriangleinclonepM56fsXhoIindicatesthepositionoftheframeshiftmutation.Wild-typeHBVclonesaredepictedatthebottom:pVPS(nucleotides2512-217)contains3'truncatedpreS/SDNA;pHBS(nucleotides2434-833)containsthefull-lengthpreS/Ssequence.Theautoradiogramshowstrans-activationofpSV2CATexpressionbypreS/S-containingtestplasmidsintransientlytransfectedCCL13cells.ThefactorofactivationwascalculatedinrelationtotheacetylationobtainedbythepUC19control.CM,unreactedchloramphenicol;1-Ac-CM,1-acetylchloramphenicol;3-Ac-CM,3-acetylchloramphenicol.vcv,tCQcC)E-U)-CXIEsIM.mnpreSIS2sF..Proc.Natl.Acad.Sci.USA87(1990)I-i"Ir,*1I7!;;.-7s( MedicalSciences:Caselmannetal.APSV2C-SV40DcatrNhiprotectedfragmentrhbo-Prlooehybridiza~tion+RNasedligestI~~~~~II~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~IProc.Natl.Acad.Sci.USA87(1990)2973PvuIIB12345190#180*150nts.1600147"'*-4150298nts.A_-PvwIlFIG.4.RNaseprotectionanalysisofCATmRNA.(A)Schematicillustration.InvitrotranscriptionofEcoRI-digestedplasmidpSPTKCAT(29)bySP6polymerasegivesrisetoanantisenseCATriboprobeof298nucleotides(nts).Hybridizationtothe5'portionofCATmRNAinitiatingattheSV40earlypromoterofpSV2CATresultsina150-nucleotideRNase-resistantfragment.TK,thymidinekinase.(B)Autoradiographshowingtherelativeincreaseinintensityofthe150-nucleotideCATfragmentaftercotransfectionofpreS/ScomprisingsubclonespM1(lane3),pM27(lane4),andpM56(lane5)isabout8-foldwithrespecttothepUC19control(lane2).Lane1,radiolabeledmolecularsizemarkers(innucleotides).CATmRNAasreflectedbytheintensityofthe150-nucleotideRNase-resistantband(Fig.4).The8-foldincreaseofCATmRNAasdeterminedbydensitometrycorrespondedtoa7-to8-foldactivationinCATassays.Wethereforeconcludethattrans-activationbyintegratedpreS/Sse-quencesoccursatthetranscriptionallevel.SincebothSgenepromoterswerecontainedinalltrans-activatingclones,weperformedRNaseprotectionmappingofpreS/S-specificmRNAstodeterminethetranscriptionstartsites.AnalysisofmRNAisolatedfromCCL13cellstransfectedwithpM27orpM56revealedtwoprotectedRNAsinitiatingattheS1promoter(Fig.5).Onetranscriptmostlikelystartedatthepreviouslydescribedinitiationsite(20)andasecondonestarted

4 about30nucleotidesupstream.Transcriptsar
about30nucleotidesupstream.TranscriptsarisingfromtheS2promoterwerenotdetected.TransientExpressionofaViral-CellularFusionTranscriptbypMlDNA.NorthernblottingoftotalandpolyadenylylatedRNAisolatedfromCCL13cellstransientlytransfectedwithpM1DNAshowedadistincttranscriptof2.5kbthathybrid-izedtototalHBV(Fig.1B,lane1)andpreS/S(Fig.1B,lane2)butnottoXDNA(Fig.1B,lane3).Thesizeofthetranscriptsuggestedaread-throughfromviraltoflanking3'ApreSlS2SOFcellularsequencesandprobablyterminationatasequencedpoly(A)siteidentified"s1700bpdownstreamoftheviral-cellularjunction.Trans-ActivationbyClonedWild-TypeHBVpreS/SSe-quences.Totestforthetrans-activationalpotentialofthecorrespondingclonedwild-typeHBVsequences,plasmidpVPSwasconstructed.ItcomprisestheHBVnucleotides2512-217(i.e.,thecompletepreSregionandtheSsequencestruncatedatexactlythesamepositionasintheintegratedHBVDNAinpM56).WhentestedforCATactivity,pVPSexerteda5-to8-foldactivatingeffectthatequaledthelevelofstimulationbytheintegratedcounterpart(Fig.3).Incontrast,clonedfull-lengthwild-typepreS/Ssequencescon-tainedinclonepHBS(nucleotides2434-833)didnotstimu-lateCATgeneexpression(Fig.3;refs.6and30).Thus,evenintheunintegratedstate,thepreS/Sgeneexertsatrans-activatingfunctiononlywhen3'truncated.DISCUSSIONWeprovideevidenceforthepresenceofapreviouslyunre-portedtranscriptionaltrans-activatorwithinthepreS/Sre-gionofHBV.Thesingle-copyHBVDNAinsertofahumanB12345ITIDcJpM564_y-SPItranscriptsrIISP2transcriptsIIIIIIIIIIpGEM3Z56riboprobeprotectedFragnents650V_.620b-750#622v527750nts.-w404650nts.620nts.FIG.5.RNaseprotectionanalysisofpreS/SmRNA.(A)Schematicillustration.Theblunt-endedinsertofplasmidpM56(nucleotides1764-1776and2703-218)wasclonedintotheSmaIsiteofplasmidpGEM3Z(Promega)generatingplasmidpGEM3Z56.InvitrotranscriptionofEcoRI-digestedplasmidpGEM3Z56bySP6polymerasegivesrisetoanantisensepreS/Sprobeof750nucleotides.Hybridizationtothe5'portionofpreS/SmRNAsresultsinRNase-resistantfragmentsof620and650nucleotides(nts).(B)Autoradiograph.ThepresenceoftwoprotectedRNAfragmentsof620and650nucleotidesfromCCL13cellstransfectedwithpM27(lane2)andpM56(lane3)indicatesthattranscriptionofpreS/SsequencesinitiatedattheknownandanadditionalunreportedstartsiteoftheS1promoter.Lanes:1,pUC19control;4,1000cpmofpreS/Santisenseriboprobe(750nucleotides);5,radiolabeledmolecularsizemarkers(innucleotides).01+hybridizationIRNasedigest-d.1,::.:i. 2974MedicalSciences:Caselmannetal.HCCwascloned.Theclone,pMl,containstruncatedpreS/SandXsequences.Amajordeletionof905bp(HBVnucleo-tides1797-2702)ledtotheeliminationoftheCgene.TheentireclonedpM1DNAwasshowntostimulatetransientexpressionoftheCATgene.Bysubcloning,itwasdemonstratedthatthestimulatoryeffectwasnotencodedwithintheXORF.A1-bpdeletion(HBVnucleotide1725)givesrisetoaframeshiftwherebyafunctionalXproteincannotbeexpressed.ThisisthelikelyreasonforthelackofactivityoftheXsequences.Ontheotherhand,stimulationofpSV2CATexpressioninsubclonepM56,whichcontained522bpofthepreSand63bpoftheSORF,wascomparabletothatofpM1.ThepreS/SregionofHBVsequencesis,therefore,sufficientforthestimulatoryeffect.TheHBVpreS/Sregionhasrecentlybeenreportedtocontainanenhancer-likeelement(31).Itwas,therefore,arguablethattheobservedstimulationmightbeduetoacis-actingeffectmediatedbythelinkageoftestandreporterplasmidduringcotransfection.Whentheenhancerlessre-porterplasmidpSV1CATwascotransfectedwiththetestplasmids,nostimulationwasobserved.Furthermore,theeffectwasabolishedwhenpM56fsXhoI,whichbearsaframe-shiftmutationwithinthepreS2ORF,wasused.Takentogether,thesedatasuggestthattheCATexpressionisstimulatedintransbyatrans-activatorproteinencodedwithinthepreS/SregionandthatthepreS2portionisnecessaryfortheeffect.RNaseprotectionanalysisofCATmRNArevealedthattrans-activationoccurredattheleveloftranscription.Tran-scriptionofpreS/S-specificmRNAstartedexclusivelyattheS1promoter.TheRNase-resistantfragmentof620nucleo-tides(Fig.5)correspondstoatranscriptpossiblystartingatapreviouslydescribedinitiationsite(20);thesecondpro-tectedfragmentof-650nucleotidesrepresentsamRNAthatoriginatesatanadditionalstartsiteclosetotheTATAboxoftheS1promoter.TheS2promoteratwhichthemajorityofStranscriptsinitiate(32)wasnotactive.Thismaybeduetoapointmutation(HBVnucleotide3118)inthepartoftheS2promoterregionthathasrecentlybeenshowntoactasarepressorelementforS1transcription(33).Inactivationofthiselementbymutationmayresultinanalteredpromoterusage.Alternatively,theswitchingoftranscriptionfromtheS2totheS1promotermaybeduetocelltype-specificpropertiesofCCL13cellsortothetruncationeventitself.NorthernblotanalysisofcellularmRNAisolatedfrompMl-transfectedCCL13cellsshowedapreS/S-specifictranscriptof2.5kb.InitiatingattheS1promoter,thistranscriptcouldterminateatapoly(A)sitethatwasidentifiedinthe3'flankingcellularsequence-1700bpdownstreamoftheviral-cellularju

5 nction.Computer-assistedtranslationofthe
nction.Computer-assistedtranslationofthehybridtranscriptrevealedastopcodon1aminoaciddownstreamoftheviral-cellularjunction.ThepreS/SsequencesofHBVhaveessentialbiologicalfunctions,includingtheregulationofvirusassembly,theinvolvementinvirusattachmenttothehepatocyte,andtheregulationofthegeneticrestrictionoftheimmuneresponse(34,35).Accordingtoourresults,thedissociationof3'sequencesassignsanewlygeneratedtrans-activatingfunc-tiontotheHBVpreS/Sregion.Duringvirusreplication,atruncatedSproteinwithtrans-activationalpropertiescouldbeencodedbyasplicedtranscript.SuchtranscriptshavebeenrecentlyreportedforHBV(36,37).Alternatively,aposttranslationalprocessingbyproteasescouldgeneratethetrans-activatorprotein.However,itisnotknownifthistrans-activatorisexpressedandifitisrelevantinthevirallifecycle.Indeed,suchafunctionmayonlybegeneratedasaconsequenceof3'truncationoftheSgeneduringgenomicintegration.ThishypothesisissupportedbytheresultsofourstudiesonanindependentlyclonedHBVinsertthatisderivedfromtheHBsAg-positivehumanhepatomacelllinehuH-4(38).WithintheintegratedHBVDNA,preS/Sse-quencestruncatedatposition219stimulateCATgeneexpressionabout14-foldinCCL13cells(39).Severalinstancesof3'truncatedpreS/SsequenceshavebeenidentifiedinvariousHCCsorhepatomacelllines(40-42);thus,trans-activationbyintegratedtruncatedpreS/SsequencesmayhaveimplicationsintumordevelopmentinHBV-infectedhepatocytes.HCCtissueMlwasagenerousgiftfromDrs.Y.WangandZ.Ma(Shanghai,People'sRepublicofChina).WearegratefultoDr.P.Herrlich(Karlsruhe)fortheriboprobevectorpSPTKCAT.TheauthorswishtothankMs.MarlenePavlik,Ms.IreneDick,andMs.ChristineSchommerforexcellenttechnicalassistance.ThisworkwassupportedbyGrantW21/86/HolfromtheDeutscheStiftungfurKrebsforschungandaResearchFellowshiptoW.H.C.fromtheDeutscheForschungsgemeinschaft(GrantCA113/1-1).1.Beasley,R.P.(1988)Cancer61,1942-1956.2.Tiollais,P.,Pourcel,C.&Dejean,A.(1985)Nature(London)317,489-493.3.Dejean,A.,Bougueleret,L.,Grzeschik,K.H.&Tiollais,P.(1986)Nature(London)332,70-72.4.Jones,N.C.,Rigby,P.W.J.&Ziff,E.B.(1988)GenesDev.2,267-281.5.Spandau,D.F.&Lee,C.H.(1988)J.Virol.62,427-434.6.Twu,J.S.&Robinson,W.S.(1987)J.Virol.61,3448-3453.7.Zahm,P.,Hofschneider,P.H.&Koshy,R.(1988)Oncogene3,169-177.8.Wollersheim,M.,Debelka,U.&Hofschneider,P.H.(1988)Oncogene3,545-552.9.Southern,E.M.(1975)J.Mol.Biol.98,503-517.10.Feinberg,A.P.&Vogelstein,B.(1983)Anal.Biochem.132,6-13.11.Chen,E.J.&Seeburg,P.H.(1985)DNA4,165-170.12.Chang,R.S.(1954)Proc.Soc.Exp.Biol.87,440-443.13.Nelson-Rees,W.A.,Daniels,D.W.&Flandermeyer,R.R.(1981)Science212,446-452.14.Graham,F.L.&vanderEb,A.J.(1973)Virology52,456-467.15.Iverson,P.L.,Mata,J.E.&Hines,R.N.(1987)BioTechniques5,521-524.16.Melton,D.A.,Krieg,P.A.,Rebagliati,M.R.,Maniatis,T.,Zinn,K.&Green,M.R.(1984)NucleicAcidsRes.12,7035-7056.17.Gorman,C.M.,Moffat,L.F.&Howard,B.H.(1982)Mol.Cell.Biol.2,1044-1051.18.Ono,Y.,Onda,H.,Sasada,R.,Igarashi,K.,Sugino,Y.&Nishioka,K.(1983)NucleicAcidsRes.11,1747-1757.19.Galibert,F.,Mandart,E.,Fitoussi,F.,Tiollais,P.&Charnay,P.(1979)Nature(London)281,646-650.20.Siddiqui,A.,Jameel,S.&Mapoles,J.(1986)Proc.Natl.Acad.Sci.USA83,566-570.21.Cattaneo,R.,Will,H.,Hernandez,N.&Schaller,H.(1983)Nature(London)305,336-338.22.Shaul,Y.,Rutter,W.&Laub,0.(1985)EMBOJ.4,427-430.23.Treinin,M.&Laub,0.(1987)Mol.Cell.Biol.7,545-548.24.Kaneko,S.&Miller,R.(1988)J.Virol.62,3979-3984.25.Koshy,R.,Koch,S.,FreytagvonLoringhoven,A.,Kahmann,R.,Murray,K.&Hofschneider,P.H.(1983)Cell34,215-223.26.Persing,D.H.,Varmus,H.E.&Ganem,D.(1987)J.Virol.61,1672-1677.27.Persing,D.H.,Varmus,H.E.&Ganem,D.(1986)Science234,1388-1391.28.Eble,B.E.,MacRae,D.R.,Lingappa,V.R.&Ganem,D.(1987)Mol.Cell.Biol.7,3591-3601.29.Angel,P.,Imagawa,M.,Chiu,R.,Stein,B.,Imbra,R.J.,Rahmsdorf,H.J.,Jonat,C.,Herrlich,P.&Karin,M.(1987)Cell49,729-739.30.Seto,E.,Yen,T.S.,Peterlin,B.M.&Ou,J.H.(1988)Proc.Natl.Acad.Sci.USA85,8286-8290.31.De-Medina,T.,Faktor,0.&Shaul,Y.(1988)Mol.Cell.Biol.8,2449-2455.32.Ou,J.&Rutter,W.J.(1985)Proc.Natl.Acad.Sci.USA82,83-87.33.Bulla,G.H.&Siddiqui,A.(1989)Virology170,251-260.34.Neurath,A.R.&Kent,S.B.(1987)Adv.VirusRes.34,65-142.35.Pontisso,P.,Petit,M.A.,Bankowski,M.J.&Peeples,M.E.(1989)J.Virol.63,1981-1988.36.Saito,I.,Oya,Y.&Shimojo,H.(1986)J.Virol.58,554-560.37.Su,T.S.,Lai,C.J.,Huang,J.L.,Lin,L.H.,Yauk,Y.K.,Chang,C.C.,Lo,S.J.&Han,S.H.(1989)J.Virol.63,4011-4018.38.Huh,N.&Utakoji,T.(1981)Gann72,178-179.39.Kekule,A.S.,Lauer,U.,Meyer,M.,Caselmann,W.H.,Hofschneider,P.H.&Koshy,R.(1990)Nature(London)343,457-461.40.Berger,I.&Shaul,Y.(1987)J.Virol.61,1180-1186.41.Imai,M.,Hoshi,Y.,Okamoto,H.,Matsui,T.,Tsurimoto,T.,Matsu-bara,K.,Miyakawa,Y.&Mayumi,M.(1987)J.Virol.61,3555-3560.42.Nagaya,T.,Nakamura,T.,Tokino,T.,Tsurimoto,T.,Imai,M.,Mayumi,T.,Kamino,K.,Yamamura,K.&Matsubara,K.(1987)GenesDev.1,773-782.Proc.Natl.Acad.Sci.USA87(1990