Molecularbiologyof Humant-Lymphotropicretroviruses

[CANCER RESEARCH (SUPPL.) 45, 45395-45445, September1985]

MolecularBiologyof HumanT-LymphotropicRetroviruses

Flossie Wong-Staal, Lee Ratner, George Shaw, Beatrice Hahn, Mary Harper, Genoveffa Franchini,and Robert Gallo

LaboratoryofTumorCellBiology,NationalCancerInstitute,Bethesda,Maryland20205

Abstract 7),a recentlyrecognizeddiseasecharacterizedbyopportunistic infectionsasa resultofsevereimmunosuppressionandOKT4 Thegenericnamefora familyofhumanT-Iymphotropicretro helperT-celldepletion(8).Consistentwithdifferencesintheirin virusesisHTLV.Two of thethreemembersinthisfamilyhave vivodiseasespectrums,HTLV-Iand HTLV-IItransformT-lym beenlinkedetiologicallyto humandiseases:HTLV-lwith adult phocytesin vitro,whileHTLV-lIlis highlycytopathicandhasno T-celIleukemiaandHTLV-Illwiththeacquiredimmunodeficiencyapparenttransformingactivity.However,allthreevirusgroups syndrome.InadditiontotheirT-celltropismanda numberof shareat leastthesecommonproperties:tropismforT-lympho othercommonbiologicalandbiochemicalproperties,themost cytes;somedegreeof cytopathyfor the infectedcells;induction uniquecommonfeaturesof thesevirusesfroma molecular of multinudeatedgiantcellsandformationofsyncytia;aMg@@ biologicalpointofviewarethepresenceofthex-Iorgenetowards preferringreversetranscnptaseofhighmolecularweight;a small the 3' end of the genomeand the phenomenonofa virus majorcoreprotein(p24);commonepitopesofgagandenvelope inducedtrans-actingfactorin activationof transcriptioninitiated proteins;anda likelyAfricanorigin. in the virallongterminalrepeat.Thesefeaturesmaynot onlybe Additionalparametersdistinguishthe HTLVfamilymembers key in understandingthemechanismoftransformationorcell fromotherretroviruses.Almostallexogenous,pathogenicanimal killingbytheseviruses,buttheyalsoprovideabasisfornew retrovirusesbelongtooneof two categories.Thechronicleu classificationofretroviruses.Inspiteofthesesimilaritiesamong kemiavirusesarereplicationcompetent,requirealonglatency HTLV-I,-II,-III,andbovineleukemiavirus,thegenomeofHTLV periodfordiseaseinduction,andlacktransformingactivityin Ill is onlydistantlyrelatedto theseotherviruses.Instead,it vitro.Theirgenomescontainonlythethreestructuralgenes(gag, showsgreaterhomologytomembersofthe Lentivirusfamily. p0!, and env) that are necessary for virus replication. The acute Therefore,allthesevirusesmayhavea commonprogenitor. leukemiaorsarcomavirusesareusuallyreplicationdefective, TwoothersalientfeaturesarosefromtheanalysesofHTLV inducediseaserapidlyinvivo,andtransformappropriatetarget III and acquiredimmunodeficiencysyndrome.(a)HTLV-IIIfre cellsefficientlyinvitro.Theyalsocarrya cell-derivedgenewhich quentlyinfectsthebrainof acquiredimmunodeficiencysyndrome codesfor a productnecessaryforthe initiationandusuallyalso patientswhosufferfromcentralnervoussystemdisorders.This maintenanceof the transformedphenotype.Membersof the notonlyidentifiesHTLV-Illasthedirectcandidateinthesecentral HTLV familyform a thirddistinctcategory.They are replication nervoussystemdisordersbutalsoposestheproblemofcrossing competent,theirgenomescontainingallthreereplicativegenes. the blood-brainbamerin therapystrategiestoeradicatethe In addition,thereare nucleotidesequencesbetweenthe env virus.(b) DifferentHTLV-lllisolatescomprisea spectrumof geneandthe3' LTRwhichcontainalongopenreadingframe relatedviruses,withthedegreeofdivergencevaryingfromvirtual (x-Ior)(9).Thex-Iorsequencesarenotderivedfromconserved identityto 10â€”15%difference.Themostdivergentregionresides cellulargenesincontrasttoretroviraloncogenes.Bothsubge intheenvelopegene.Whetherthisfindinghasimplicationsinthe nomicx-IormRNAand the actualx-Iorproteinproducthave developmentofan effectivevaccineforacquiredimmunodefi beenidentifiedinthe casesof HTLV-Iand-II.Therecentobser ciencysyndromeremainstobedetermined. vationthattranscriptioninitiatingwithintheviralLTRisgreatly enhancedin the homologousHTLV-infectedcells(10, 11) has Introduction implicatedthex-Iorproteinasthe mediatorofthisTATphenom Allhumanretrovirusesisolatedandcharacterizedtodateare enon.OtherretrovirusesthatalsoexhibitTATandcontainthe T-Iymphotropic,especiallyforthe OKT4-positivehelperT-cell. x-IorgenearethedistantlyrelatedBLV(12),simianretroviruses Thefirstsuchviruseswereobtainedfrompatientswithmature (STLV-I)whichare doselyrelatedto HTLV-l,3and possibly T-ceIIcancersandwerenamedHTLV.2Threedistinctsubtypes membersofthe Lentivirusfamily.4 havenowbeenidentified.HTLV-Iistheetiologicalagentofadult T-cellleukemia,a diseaseendemicin southwesternJapan,the GenomesofHTLV-landHTLV-II Caribbean,CentralandSouthAmerica,andAfrica(1, 2). HTLV IIwasfirstisolatedfroma patientwithhairycellleukemia(3)but The completenucleotidesequenceofoneisolateofHTLV-I hasnotbeenlinkedtoanyhumandisease.Morerecently,athird (13) and partialsequencesof other HTLV-I isolates(14, 15) as memberof this family, HTLV-Ill, has been shown to be the wellasoneofHTLV-lI(16â€”18)havebeendetermined.Themost etiologicalagentof theacquiredimmunodeficiencysyndrome(4â€” unexpectedfeaturerevealedfrom these studiesis a regionof about1.6 KB betweenthecarboxyterminusofthe envgene 1 Presented at the HTLV Symposium, December 6 and 7, 1984, Bethesda, MD. and3' LTRthatcontainsseveralopenreadingframes(Chart1). 2 The abbreviations used are: HTLV, human T-ceII leukemia/lymphotropic virus; Thisregion,initiallyreferredto as the pX region(13), is not p24, proteinwith a molecularweightof 24,000 (otherproteinsdefinedsimilarly); LTR, longterminalrepeat;TAT, trans-actingtranscriptionalactivation;BLV,bovine closelyhomologoustocellularsequencesofvertebratecells(19) leukemiavirus;KB, kilobase;AIDS, acquiredimmunodeficiencysyndrome;ARC, acquired immunodeficiencysyndrome-related complexes; ATL, adult T.cell leuke 3 M. Voshida, personal communication. mia. 4 J. Clements, personal communication.

CANCERRESEARCHVOL.45 SEPTEMBER1985 45395

The Genome of HTLV-I and -Il betweenthetwo transformingHTLVs.Studieson the promoter enhanceractivityof LTRsof HTLV-Iand HTLV-IIshowedthat 5' LTR pX 3' -LTR - GAG Non-coding P01 ENV @â€”@-â€”â€”â€”â€˜@ transcriptionofLTR-linkedgenesiselevatedmanyfoldininfected c=* I [ I T@t LOR4J cells,indicatingatrans-actingtranscriptionalactivationphenom p63 p100? p@ p42 enonviaa viralprotein(10, 31).Thereisindirectevidencethat

Â£!!p24 thex-!orproteinisresponsibleforthisenhancingactivity.Based -@@! I onthesestudies,onecanconstructamodelinwhichthex-!or @@ I I I t I I @!@Ã§.!! proteincanalsoregulatecellularpromoter-enhancerfunctionat icx@ distantsites,and in whichthe cellulargenesregulatedare Chart 1. The genomes of HTLV-I and HTLV-II. The genetic structure of HTLV-I and the predicted protein products are shown. GAG, core antigens; POL, DNA associatedwithT-cellproliferation.Thereforethemechanismof polymerase (reverse transcriptase); ENV, envelope glycoprotein; NCR, noncon transformationby HTLV is more similarto certain DNAtumor served region; LOR, long open reacing frame. HTLV-II genome is very similar to virusesthanto otherretroviruses.Thereare alreadyseveral HTLV-I,excepta proteasegeneis situatedbetweengag andpolgenes whereit is indicated noncoding' for HTLV-I, and the long open reading frame protein of a genesidentifiedthat are activatedby infectionwith HTLV-Iand p38. HTLV-Il,andallthesehavebeenlinkedto T-cellproliferation(32, 33). Therefore it is directlytestable whether the x-!or protein andthereforeisnota typicalretroviraloncogene.Deletionsand bindsto the enhancer-promotersequencesofthesegenes. substitutionswithin the first 0.6 KB of pX found in a variant, Unliketheinvitrotransformedcells,freshATLcellsfrequently HTLV-Ib,obtainedfromanAfricanATLpatientdidnotaffectthe contain no detectableviral mRNA or proteins,includingx-!or transformingcapacityof this virus (15, 20, 21). Comparisonof (34). In addition,althougheachof theseATL cellsamplesis thepXsof HTLV-IandHTLV-lIrevealedthatthefirst0.6 KBis monoclonal,theintegrationsites of these provirusesvary(35). nonconservedbutthatthefollowing1.0 KBishighlyconserved Sothevirusdoesnotseemto functionincisor transinmain andcontainsasinglelongopenreadingframeX or x-Ior(16, tamingthe leukemicstate.We speculatea two-stageleukemo 22). Spliceacceptorconsensussequencesare preserit5' to x genesisevent, with the early stage beinganalogousto the in br whichdoes not initiatewithan ATG codon(16). Recent vitro transformationprocess, resulting in immortalizationand evidencesuggeststhat the initiationATGcodonis derivedfrom enhancedproliferationoftheinfectedcells.Thisinturnincreases the env gene by a doublesplicingevent.5The HTLV-lx-Ior thechancefora mutationorgenerearrangementwhichleadsto sequencepredictsa proteinproductwith a molecularweightof theprogressionofthedisease.Atthislatestage,thevirushas at least38,000.SeraofpatientspositiveforHTLV-lrecognizea doneits damageandis no longerneeded. Mr 40,000-42,000 protein expressed in all HTLV-l-transformed cells,includingnonproducercellsthat do not synthesizevirusor virionstructuralproteins(23).Aminoacidanalysisofcyanogen GenomeofHTLV-IlI,theEtiologicalAgentof AIDS bromidecleavagefragmentsof this protein(24)as wellas direct bindingofthisproteinbyantibodyraisedagainstpeptidessyn A novel memberof the HTLV familydesignatedas HTLV-IlI thesizedfromthe predictedprimarysequence(25â€”27)indicate hasrecentlybeenidentifiedastheetiologicalagentof AIDS,a thatp42isat leastinpartcodedbyx-Ior.Sequenceanalysisof diseasecharacterizedbydepletionof the OKT4-positivehelper theBLVgenomealsorevealsthepresenceofanopenreading T-ceIIs(36).We haveclonedthe genomesof severalisolatesof framesimilarinsizeto HTLV-lx-Iorinthecorrespondingregion HTLV-lll (37, 38). Nucleotidesequenceanalysisof two of these (28). clonesrevealedacompletegenomeof9213 BP,withfivelong openreadingframesflankedbyLTRsequences(39,40)(Chart 2). The LTR is 634 nudeotideslong.The U3, A, and U5 regions PossibleMechanismofTransformationandLeukemogenesis are 453, 98, and83 nucleotideslong,respectively.ThetRNA by HTLV-IandHTLV-Il bindingsitewas determinedtobe [email protected] virusknowntoutilizeatRNA@asprimeristhemousemammary Cells infectedby HTLV-l and HTLV-llin vitroare initially tumorvirus. polydonaland go througha growth crisis within 4â€”6wk.Pre The first open readingframepotentiallycodesfor a gag dominantlycionalcellsthenemergeasimmortalizedcells(29). precursorof 512 aminoacids. Actual alignmentof the amino OftenthesecellsbecomeindependentofT-ceIIgrowthfactor acidsequenceofthefirst 15 residuesofthemajorcapsidprotein and morphologicallyresembletheprimaryleukemiccells.Al (p24) with the sequence in this region predicted an amino though the transformedcells are donal, the site of provirus terminalpeptide of 132 amino acids.6This protein, probably integrationisvariablein differentclones(30). The lack of a correspondingto a p17 seen in disruptedHTLV-IIlviruses,is conservedintegrationsitesuggeststhatthesevirusesactbya similarinsizeto theamino-terminalgagproteinofHTLV-I(126 transmechanismincellulartransformation;,e., a diffusibleviral aminoacids).Furthermore,theimmediatejuxtapositionofthe proteinproductisdirectlyinvolved.Byanalyzingexpressionsof p24 protein with the amino-terminalp17 and lack of a small differentviral structuralproteinsat differenttime intervalsafter phosphoproteininterpolatingbetweenthese two protiens are infectionandclonesoftransformedcellscontainingonlydefec analogoustothestructureofthegaggeneproductsofHTLV-l, tive proviruses,we have ruledout the expressionof gag, p0!, -Il, and BLV and differ from murine,avian, feline, and primate andenvproteinstobe necessaryforthe initiationoftransfor type C and type D retroviruses. mation(29).The onlyremainingcandidateisthex-Iorprotein, Thesecondlongopenreadingframerevealsnumerousregions especiallyinviewof the highdegreeof conservationofthisgene of aminoacidsimilaritytothepo!geneproductsofotherretro

5 A. Aldovini, et a!., unpublished observation. 6 F. Wong-Staal, et al., unpublished data with those of S. Oroszlan.

CANCERRESEARCHVOL.45 SEPTEMBER1985 45405

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1985 American Association for Cancer Research. HTLVMOLECULARBIOLOGY @IIâ€˜@- ..jIIJJJIII!iiiiiiiiiiiiiiiirn.ii @@iii@ii.@ii iiui@iiiiiiiiii@ i @ IIIIIII@II11111111IIII0III@11111liiIIIIiIiIliiiIIIII @IIUI@III III @IIIIOI@lIIII@IIII@liiiIII @ IIIiiirrriiiiiitiaii . @@1@1E1i1i1iIIMâ€• D III III I I III A III tillENV Ill@f@fl@ff

uu1@1uI@I ,I,,lu,lI 1,111,11, uu!J1It@I II,I@,,lI 111111111 I!l@@1llI IIIl@uIII ,III@,luJ

1000 2000 3000 4000 5000 6000 7000 8000 9000 Chart 2. Genetic structure of the HTLV-lll genome. See legend to Chart 1 for notations. In addition, SOR, short open reading frame; 3' OAF, 3' open reading frame.

viruses.Basedon thesehomologies,weconcludedthatthis of virusparticlesmorphologicallyindistinguishablefromHTLV openreadingframeencodesthep0! genewhichyieldsthree III, and the steady increaseof viral DNA and RNA sequences products:aproteaseatits5'-terminus;areversetranscriptase detectableinthe transfectedcellsup to 14 days.Moreinterest inthemiddle;andanendonucleaseatits3'-terminus.Although ingly,cellstransfectedwithHTLV-IIIspecificallyshowedadra HTLV-Idoesnot encodea functionalproteaseinthisregion, maticdecreaseincellnumberafter14 days.At 18 days,none HTLV-IlandBLVdo. oftheremainingcellshadevidenceofvirus.Theseobservations The thirdopen readingframe(sor)is relativelyshortand suggestthattheclonedHTLV-IIIgenomewasbiologicallyactive capableofencodingaproteinof203aminoacids.Thisisfollowed inproductionofvirionsthatarebothinfectiousandcytopathic. bya sequenceof584 BPcontainingmultiplestopcodons.There Constructionof specificmutantsof this done will allowfurther is nodirectevidencethatthisopenreadingframeisfunctional. dissectionofthe cytopathicdeterminantsofthis virus. ExpressionofrandomfragmentsoftheHTLV-lllgenomeinopen readingframevectorsshowedthatpeptidesexpressedfromthis Stateof theViralDNAin InfectedCells regionarenot cross-reactivewiththe patient'ssera(41). The fourthopenreadingframe(env)encodesaprotein863 HTLV-IlIis a completelyexogenousvirus,lackinganycell aminoacidslong.Thepredictedproductresemblestheenvelope derivedsequencesasdemonstratedbythelackofhybridization precursorproductsofotherretroviruses,includingahydrophobic ofitsgenometouninfectedcellDNA(37,38).Twocomponents regionin the middleprecededbyan arginine-richhydrophilicofviralDNAexistintheinfectedcells;oneexistsaspolyclonally regionwhichcontainsthe cleavagesite of env precursorsinto integratedprovirus,the other,as unintegratedcircularor linear exteriorandtransmembraneproteins.Inaddition,ashorthydro viralDNA.Thepolyclonalpatternofintegrationisexpectedfora phobicsequenceatthe aminoterminusresemblessignalpep viruswhichiscytopathicanddoesnotcauseadonalproliferation tidesof env precursorproteins,andthe regionofthe protein of the infectedcells,in contrastto HTLV-Iand HTLV-lI.The whichliesbetweenthisputativesignalpeptideandtrans-mem persistenceofa largeamountof unintegratedviralDNA is braneproteinishydrophilicandcontains24potentialasparagine unusualforretrovirusesbuthasbeendescribedforothercyto linkedglycosylationsites.However,thepredictedproductofthis pathicretroviruses,suchas spleennecrosisvirus(44), some openreadingframediffersfromenvproteinsofotherretroviruses strainsofavianleukosisvirus(45), andvisnavirus(46).This bythepresenceofanadditional180aminoacidsatthecarboxyl featurecanthusbe consideredtobe a hallmarkofcytopathic terminus. retroviruses. The fifthopenreadingframe(3' oil) hasa maximalcoding capacity of 216 amino acids but is polymorphicand slightly DetectionofHTLV-IllDNAandRNAinFreshTissues truncatedinsomeproviruses.Itisencodedbyanabundant2.0- KB mRNA whichalsocontains414 nucleotidesfromthe 3' env We haveexamineddoseto 100 freshtissuespecimensfrom geneaswellassequences5'ofgagand3' ofsorasa resultof patientswithAIDSorARCforthepresenceofHTLV-IIIDNAby a doublesplicingevent(42).SincetranscriptionutilizingHTLV Southernhybridization,and many of these tissues were also Ill LTR is trans-activatedininfectedcells(11), a phenomenon examinedfor viral RNA expressionby in situ hybridization. thatisbelievedtobemediatedbythex-!orproteinsintheHTLV Althoughbyserologyandactualvirusisolationgreaterthan90% I, -II, andBLVsystems,aproteinproductencodedbythe2.0- of theseindividualswerepositivefor HTLV-lll,onlya small KB mRNA may havea functionanalogousto x-!or. percentage(@cz20%)ofthesespecimenshaddetectableviralDNA or RNAsequences.Moreover,eventhe positiveDNAsamples BiologicalActivity of Cloned HTLV-lllGenome yieldedbandsof very low intensity,suggestingthat only a rare cell in the populationis infected.This is supportedby in situ We haveusedtheprotoplastfusiontechniquetointroducea hybridizationwhich revealeda rare positivecell amongmany completeHTLV-lIIclone into normalcord blood lymphocytes cellsnotexpressingviralRNA(Fig.1). Roughlythesamepro (43).Virusproductionwasevidencedbytheexpressionofp24 portionof ARCpatientsasAIDSpatientswaspositivefor HTLV andp15 inupto 10% ofthetransfectedcellpopulation,release III, and viralsequenceswere detectedmorecommonlyinlymph

CANCERRESEARCHVOL.45 SEPTEMBER1985 4541s

cantchangesintheaminoacidsequenceoftheenvelopeprotein and consequentlyalter the immunologicalcross-reactivityof differentHTLVisolates. .. Discussion HTLV isa retrovirusfamilywith manyuniquefeatures.It isthe firsthumanretrovirusidentifiedandthefirsttobelinkedetiolog icallyto humandiseases.Thesevirusesshow exquisitetarget cell specificityfor the T4-positivehelperT-Iymphocytebothin I vivoandinvitro,leadingtouncontrolledproliferation(cancer)or prematureterminationofgrowth(immunosuppression)ofthese cells.Thesediseasesystems,namelyadultT-cellleukemiaand acquiredimmunodeficiencysyndrome,as wellas the possibility -L@ to reproducesomeaspectsofthediseasesinvitro,providean unprecedentedopportunitytostudybasicmechanismsinvolved Fig. 1. Detection of HTLV-IlI RNA in peripheral blood mononuclearcells from a inregulationofcellgrowth. patient with lymphadenopathy by in situ hybridization 10@cpmof @S-RNAfran scribed from pBHlO-R3 containing 9 KB of the HTLV-IIIgenome in pSP64 used as ThevirusassociatedwithATL,HTLV-l,anditsdoserelative, a probe. Exposure was at 4Â°Cfor2 days. HTLV-lI,had someunusualpropertiesascomparedtoother leukemogenicretroviruses.Althoughtheydo notcarryaclassical nodes than peripheralblood. However,even lymph nodes of oncgene,theypossessagene(x-!or)whichisnotpartof the patientswith lymphadenopathycontainedfewerthanone virus normalreplicativemachineryofretroviruses.Furthermore,tran positivecellin 1000, indicatingthatthe virusdoesnot playa scÃ¨iptioninitiatedwithintheviralLTRisgreatlyenhancedinvirus directroleinthe proliferationoflymphocytesinthissyndrome. infectedcells.Thisphenomenon,referredtoas TATactivation, Similarly,noneof15 Kaposisarcomatissuesexaminedrevealed isbelievedtobemediatedbythex-!orgeneproduct. DNAsequenceseventhoughhistopathologicalexaminationin ThevirusassociatedwithAIDS,HTLV-III,isanexogenousT dicated30â€”90%tumorcells(38).Thisresultarguesthat Kaposi lymphotropicretrovirusthatsharesmanycommonpropertiesas sarcomais a secondarydevelopmentofHTLV-Illinfectionin wellasdistanthomologywiththetwopreviouslyisolatedhuman AIDSpatients. retroviruses,namelyHTLV-land HTLV-lI.In addition,cellsin In searchof a tissuereservoirforHTLV-lIIreplicationby fectedwithHTLV-lllcontainatrans-actingfactorwhichactivates surveyingmultipletissues for viral sequences,we found that transcriptioninitiatedwithinviralLTR(11).This property,called braintissueswerefrequentlypositive(47).Thisresultwasnot TAT,sofaruniquetovirusesoftheHTLV/BLVfamily,mayform dueto infiltrationoflymphocytessincelymphoidtissues(lymph thebasisforclassificationofothernewlydiscoveredviruses.In node,spleen,peripheralblood)fromthesamepatientwereeither spiteof theirsimilarities,HTLV-IIlalsoexhibitsmanydifferent lessor not detectablypositive,andsincethe histologyofthe molecularfeatures,as expectedfor its distinct biologicalattri positivebraintissuesindicatedan absenceof lymphocytes. butesandclinicalmanifestations.Thepolyclonalpatternofinte Again,this findingis supportedby in situ hybridizationanalyses, grationandpersistenceofunintegratedviralDNAin the long whichalsodemonstratethatHTLV-lllisnotonlyfoundfrequently term infectedcellsare characteristicof cytopathicretroviruses, inbraintissues,butit isalsohighlyexpressed,possiblyactively althoughthe mechanismof cytopathy is still not understood. replicatedin this tissue.Furthermore,thisanalysisshowedthat Giventheexistenceofa proteinfunctionallyanalogoustox-!or thedimensionsoftheinfectedcellswereinconsistentwiththeir whichisbelievedtobetheâ€œtransformingâ€•proteinofHTLV-land beinglymphocytes.Theprecisenatureof the virus-positivecell HTLV-II, it is possiblethat HTLV-lll has potentialtransforming in the brainwill be determinedby immunocytometrywithanti capacitywhichis maskedby the cytopathiceffect of the virus. bodiesagainstspecificcellularand viral markers.SinceAIDS Availabilityof a biologicallyactive DNA done of HTLV-lll now patientsoftenhavesubacuteandacutedisordersinvolvingthe allowsconstructionofvariousdeletionmutantsin biological centralnervoussystem(48),HTLV-Illinfectionof the brainmay assaystoaddressthemechanismofcytopathogenicityand/or havea directroleinthesedisorders. transformationbyHTLV-IIl. AlthoughHTLV-lllsequencescanbedetectedinDNAorRNA of freshtissuesofAIDSandARC patients,theyare generally HeterogeneityofDifferentHTLV-lIlIsolates presentonlyina rarecell.Suchisthecasealsoforlymphnode tissuesof patientswithlymphadenopathy,indicatingthatthe We haveover100 isolatesofHTLV-lllinourlaboratoryand virusis notdirectlyinvolvedinthe proliferationofthe lympho haveanalyzedabout20 provirusesinbothcelllinesandfresh cytes. Negativeresultswith Kapositumor tissuesalso ruleout tissuesbySouthernhybridization.Theresultsindicatethatdif thevirusashavinganimmediateroleintheirdevelopment.Of ferentisolatesofHTLV-IlIcompriseaspectrumofrelatedvi interestisthefindingoffrequentHTLV-llIinfectioninthebrains ruses.Theextentof divergencerangedfromvirtualidentityin ofpatientswithAIDS,whichmakesthisvirustheprimecandidate restrictionenzymemapsto lessthan50% correspondencein as the directfactorfor a multitudeofcentralnervoussystem cleavagesites.By heteroduplexanalysis,theregionsofgreatest disordersassociatedwithAIDS.Thereareprecedentsforretro divergencecanbe localizedtotheenvgene.However,itisnot virusesthathaveadualtropismforlymphocytesandbrain,most knownif thesenudeotidechangeswouldtranslateintosignifi notablyvisnaviruswhichcausesdegenerativeneurologicaldis

CANCERRESEARCHVOL.45 SEPTEMBER1985 4542s

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1985 American Association for Cancer Research. HTLVMOLECULARBIOLOGY easesin sheep(49). Recentdatarevealedsignificantgenome Aced. Sd. USA,81: 4617â€”4621,1984. 19. Franch@,G., Wong-Staal, F., and Gallo, A. C. Molecular studies of human T homologybetweenHTLV-lllandseveralmembersoftheLenti cell leukemia virus and adult T-cell leukemia. J. Invest. Dermatol., 83: 635- virusfamily,includingvisna,equineinfectiousanemiavirus,and 665,1984. 20. Hahn, B. H., Shaw, G. M., Popovic, M., LoMonico, A., Gallo, A. C., and Wong caprinearthritisandencephalitisvirus(50,51).Anotherparallel Staal, F. Molecular cloning and analysis on a new variant of human T-cell betweenvisnavirusand HTLV-IIIis the extentof geneticdiver leukemia virus (HTLV-lb) from an African patient with adult T cell lymphoma. gence,particularlyintheenvelope.Forvisna,geneticdriftofthe Int. J. Cancer, 34: 613â€”618,1984. 21. Shaw, G. M., Hahn, B. H., Popovic, M., LoMonico, A., wong-Staal, F., and virusoccursintheinfectedanimal,enablingthevirustoescape Gallo, A. C. Molecular cloning and analysisof different human T-IymphOtrOpiC immunesurveillanceperiodically(52). Examinationof multiple retroviruses from patients with AIDS or T-celI malignancy. In: M. J. GOttlieb isolatesfromthesameHTLV-III-positivepatientinthecourseof and J. E. Groopman (ads.), Acquired Immune Deficiency Syndrome. UCLA SymposiumonMolecularandCellularBiology,Newseries,Vol.16.NewYork: thediseaseisin progressto determineifa similarsituationexists Alan A. Use, Inc., 1985. for HTLV-lll. Whetherdivergencein the envelopeof HTLV-lII 22. Shaw, G. M., Goads, M. A., Fhckinger,G. H., Hahn, B. H., Gallo, A. C., and Wong-Staal,F.GenomesofevolutionarydivergentmembersofhumanT-cell mayposeproblemsfordevelopmentofan effecivevaccinefor leukemiavirus family (HTLV-I and HTLV-II)are highly conserved, especiallyin AIDSremainstobedetermined. pX. Proc. Nail. Aced. Sd. USA,81: 4544-4@48,1984. 23. Lee, T. H., Homma, T., Schultz, K. T., McLane, M. F., Tachibana, N., Howe, C. w. S., and Essex, M. E. Antigens expressed by human T-cell leukemia virus-transformedcells. In: A. Gallo, M. Essex, and L. Gross (ads.),HumanT Cell Leukemia/LymphomaVirus, pp. 111-120. Cold Spring Harbor, NV: Cold References Spring Harbor Laboratory, 1984. 24. Lee, T. H., Coligan, J. E., Homma, T., Sodroski, J. G., Haseltine, W. A., 1. Gallo, R. C. Human T-ceIIleukaemia-lymphomavirus and T-celI malignancies Sallahuddin, 5. z., Wong-Staal, F., Gallo, A. C., and Essex, M. Antigen in adults. In: J. Wyke and A. Weiss (ads.), Cancer Surveys, Vol. 3, pp. 113â€” expressedbythe3' terminalregionofhumanTcellleukemiavirus:evidence 159. Oxford: Oxford Wiversity Press, 1984. for a functional gene flanked by the env gene and 3' LTR. Science (Wash. 2. Broder, S. (ed). Symposium on HTLV. Cancer Res. (Suppl.), 45: 4519s- DC),220: 57â€”61,1984. 47115,1985. 25. Kiyokawa, T., Seiki, M., Imagawa, K., Shimizu, F., and Yoshida, M. Identifica 3. Kalyanaraman, V. S., Samgadharan, M. G., Robert-Guroft, M., Miyoshi, I., tion of a protein (p40â€•)encodedby a unique sequence pX of human T-cell Blayney, D., Golde, D., and Gallo, R. C. A new subtype of human T-cell leukemiavirus type I. Gann, 75: 747â€”751,1984. leukemia virus (HTLV-II)associated with a T-celIvariant of hairy cell leukemia. 26. Miwa, M., Shimotohno,K., HOShinO,H.,Fujino,M., and Sugimura,T. Detection Science (Wash. DC), 218: 571â€”573,1982. of pX proteins in human T.cell leukemia virus (HTLV)-infectedcells by using 4. Popovic, M., Samgadharan, M. G., Read, E., and Gab, A. C. Detection, antibodyagainstpeptidededucedfromsequencesofX-IVDNAofHTLV-1and isolation,andcontinuousproductionofcytopathicretroviruses(HTLV-llI)from Xc DNA of HTLV-lI proviruses. Gann, 75: 752â€”755,1984. patients with AIDS and pro-AIDS. Science(Wash. DC), 224: 597-600, 1984. 27. Wachsman,W., St*notohno, K., Clark, S. C., Golde, D. W., and Chen, I. S. V. 5. GaIlo, A. C., Salahuddin, S. Z., Popovic, M., Shearer, G. M., Kaplan, M., Expression of of the 3' terminal region of T-cell leukemia viruses. Science Haynes, B. F., Palker, T. J., Redfleld, A., Oleske, J., Safai, B., White, G., (Wash. DC), 226: 177â€”179,1984. Foster, P., and Markham, P. D. Frequent detection and isloation of cytopathic 28. Rice, N. A., Stephenson, A. M., Couez, D., Deschamps, J., Kettmann, A., retroviruses (HTLV-lll) from patients with AIDS and at risk for AIDS. Science Bumy, A., and G@den,A. V. The nucleotide sequence of the env gene and (Wash. DC), 224: 500â€”503,1984. postenv region of bovine leukemiavirus. Virology, 138: 82-93, 1984. 6. SchUPbach,J., Popovic, M., Gilden, R. V., Gonda, M. A., Sarngadharan, M. G., and Gallo, R. C. Serological analysis of a subgroup of human T-lymphO 29. De Rossi, A., Aldovini, A., Franchini, G., Mann, D., Gaflo, A. C., and Wong tropic retroviruses (HTLV-llI)associated with AIDS. Science(Wash. DC), 244: Steal, F. Clonal selection of T.lymphocytes infected by Cell-freehuman T-cell 503â€”505,1984. leukemia/IymphotropicvirustypeI:parametersofvirusintegrationandexpres 7. Samgadharan, M. G., Popovic, M., Bruch, L., SchUpbach,J., and Gab, A. C. sion.Virology,143:640â€”645,1985. Antibodies reactive with human T-IymphOtrOpICretroviruses(HTLV-III)in the 30. Wong-Staal,F.,Hahn,B.,Manzari,V.,Colombini,S.Franct*u,G.,Gelmann, serum of patients with AIDS. Science(wash. DC), 224: @06-@08,1984. E. P., and Gab, A. C. A survey of humanleukemiasfor sequencesof a human 8. Groopman, J. P. (ed). Seminars in Oncology, Vol. 11. New York: Grune & retrovirus, HTLV. Nature (Lend.),302: 626â€”628,1983. Stratton, Inc., 1984. 31. Haseltine,W. A., Sodroski, J., and Rosen, C. The lot gene and pathogenesis 9. Haseltine,W. A., Sodroski, J., Patarca, A., Briggs, D., Perkins, D., and wong of HTLV-l, -II, and -III. Cancer Res. (Suppl.),45: 4545s-4549s, 1985. Staal, F. Structure of 3' terminal region of type II human T lymphotropic virus: 32. Manzan,V., Gab, A. C., Franchini,G., Westin, E., CeCcherini-NeIIi,L,Popovic, evidencefornewcodingregion.Science(Wash.DC),225:419-421,1984. M., and Wong-Staal, F., Abundant transcription of a cetular gene in T cells 10. Sodroski, J. G.. Rosen, C. A., and Haseltine,W. A. Trans-actingtranscriptional infected with human T-cell leukemia-lymphomavirus. Proc. Nail. Aced. Sd. activation of the long terminal repeat of human T lymphotropic viruses in USA,80: 11â€”15,1983. infected cells. Science (Wash. DC), 225: 381â€”385,1984. 33. Arya, S. K., Wong-Staal, F., and Gallo, A. C. Transcriptional regulation of a 11. Sodroski, J., Rosen, C., Wang-steal, F., Salahuddin,S. Z., Popovic, M., Arya, tumor promoter and mitogen-induciblegene In human lymphocytes. Md. Cell. S., Gallo, A. C., and Haseltine,W. A. Trans-actingtranscriptional regulation of Bid., 4: 2540-2542, 1984. human T-ceIIleukemia virus type III long terminal repeat. Science(Wash. DC), 34. Franchini, G., Wong-Staal, F., and Gallo, A. C. Human T-cell leukemia virus 227: 171â€”173,1985. (HTLV-I) transaipts in fresh and cultured cells of patients with adult T-cell 12. Rosen, C. A., Sodroski, J. G., Kettman, A., Bumy, A., and Haseltine, W. A. leukemia. Proc. Nail. Aced. Sd. USA, 81: 6207-621 1, 1984. Trans-activation of the bovine leukemia virus long terminal repeat in BLV 35. Seiki, M., Eddy, A., Shows, T. A., and Voshida, H. Non-specificintegration of infected cells. Science (Wash. DC), 227: 320â€”322,1985. HTLV provirus genome into adult T-cell leukemia cells. Nature (Lend.), 309: 13. Seiki, M., Hattori, S., Hirayama, V., and Voshida, M. Human adult T-cell 640â€”642,1984. leukemia virus: complete nucleotide sequence of the provirus genome into 36. Gallo, A. C. The human T.cell leukemia/lymphotropic retroviruses (HTLV) grated in leukemiacell DNA. Proc. Nail. Aced. Sd. USA,80:3618-3622,1983. family: past, present, and future. Cancer Aes. (Suppl.), 45: 4524sâ€”4533s, 14. Josephs, S. F., Wong-Staal, F., Manzan,V., Gallo, A. C., Sodroski,J. G., Trus, 1985. M. D., Perkins, D., Patraca, A., and Haseltine, W. A. Long terminal repeat 37. Hahn, B. H., Shaw, G. M., Arya, S. K., Popovic, M., Gallo, A. C., and Wong structureofanAmericanisolateofhumanT-cellleukemiavirus.Virology,139: Steal, F. Molecular cloning and characterization of the virus associated with 334â€”345,1985. AIDS (HTLV-IlI).Nature (Lend.),312: 166â€”169,1984. 15. Ratner, L., Josephs, S. F., Starcich, B., Hahn, B., Shaw, G. M., Gallo, A. C., 38. Shaw, G. M., Hahn, B. H., Arya, S. K., Groopman, J. E.. Gallo, A. C.. and and Wong-Staal, F. NUcleOtidesequence analysis of a variant human T.cell Wong-Staal,F.MolecularcharacterizationofhumanT-ceIIleukemia(lympho leukemia virus (HTLV-lb) provirus with a deletion in pX. J. Virol., in press, tropic) virus type III in the acquired Immunodeficlency syndrome. Science 1985. (Wash. DC), 226: 1165â€”1171,1984. 16. Gelmann, E. P., Franchini, G., Manzan, V., Wong-Staal, F., and Gab, A. C. 39. Starcich, B., Rather, L, Josephs, S. F., Okamoto, T., Gallo, R. C., and Wong Molecular cloning of a unique human T.cell leukemia virus (HTLV-lI). Proc. Steal, F. characterization of the long terminal repeat sequences of the AIDS Nail. Aced. Sd. USA,81:993-997,1984. virus human T-cell IymphOtrOpiC(leukemia)virus type III (HTLV-IIl). Science 17. Shimotohno, K., Golde, D. W., Miwa, M., Sugimura, T., and Chen, I. S. V. (Wash. DC), 227: 538â€”540,1985. Nucleotide sequence analysis of the long terminal repeat of human T-cell 40. Rather, L., Haseltine,W., Patarca, R., Uvak, K. L, Starcich, B., Josephs, S. leukemiavirus,typeII.Proc.Natl.Acad.Sd.USA,81:1079â€”1082,1984. F., Doran, E. A., Rafalski, J. A., Whitehom, E. A., Baumeister, K., Ivanoff, L, 18. Sodroski, J., Trus, M., Perkins, D., Patarca, A., Wong-Staal, F., Gelmann,E., Petteway.S.A., Pearson,M.L, Lautenberger,J.A.,Papas,T.S.,Ghrayeb, Gallo, R. C., and Haseltine, W. A. Repetitive structure in the long terminal J., Chang, N. T., Gallo, A. C., and Wong-Staal, F. COmplete flucleotide repeat element of a type II human T-cell leukemia virus (HTLV-II).Proc. Nail. sequencesoftheAIDSvirus,HTLV-III.Nature(Lond.),313:277-284,1985.

CANCER RESEARCH VOL. 45 SEPTEMBER 1985 45435

41. Chang, N. T., Chandra, P. K., Bame, A. D., McKinney,S., Rhodes,D. P., Tam, 47. Shaw, G. M., Harper, M. A., Hahn, B. H., Marselle, L, Epstein, L, Oleske, J., S. H., Shearman, C., Huang, J., Chang, T. W., Gal$o,A. C., and Wong-Staal, Price, A. W., Navia, B. A., Petito, C. K., Cho, E-S., O'Hare, C. J., Groopman, F. ExpressionofE. coIl of open readingframe gene segments of type Ill human J. E., Wong-Staal,F., and Gallo, A. C. HTLV-IIIinfection of brains of demented T-cel IymphOtrOpiCvirus.Science(Wash. DC), 228: 93-96, 1985. children and adults with AIDS. Science (Wash. DC),227:177-182,1985. 42. Arya, S. K., Chan, G., Josephs, S. F., and WOng-Staal,F.Trans-activatorgene 48. Snider, W. D., Simpson, D. M., N@sen, 5., Gold, J. W. M., Merroka, C, E., of human T-lymphOtrOpiCvirustype III (HTLV-lll). Science (Wash. DC), 229: andPosner,J.B. Neurologicalcomplicationsofacquiredimmunedeficiency 69â€”73,1985. syndrome: analysis of 50 patients. Ann. Neurol., 14: 403â€”418,1983. 43. Fisher, A. M., Collalti, E., Rather, L, Gab, A. C., and Wong-Staal, F. A 49. Sigurdssen, B., Palsson, P. A., and Van Bogaert, L Pathology of visna. Acts molecular clone of HTLV-III with biological activity. Nature (Load.), in press, Neuropathol., 1: 343â€”362,1962. 50. Goads. M. A., Wong-Staal, F., Gallo, A. C., Clements,J. E., Narayan, 0., and 1985. Gilden, A. V. Sequence homology and morphologic similarity of HTLV-III and 44. Keshet, E., and Temin, H. M. Cell killing by spleen necrosis virus is correlated visnavirus, a pathogenicLentivirus.Science(Wash. DC),227:173â€”177,1985. witha transientaccumulationofspleennecrosisvirusDNA.J.Virol.,31:376- 51. Gonda, M. A., Wong-Staal, F., Gallo, A. C., Cements, J. E., and Gilden, A. V. 388,1979. HeteroduplexmappinginthemolecularanalysisofthehumanT-celleukemia @ 45. Weller, S. K., Joy, A. E., and Temin, H. M. COrrelationbetween killing and (tymphotropic)viruses.CancerRes.(Suppi.),45:4553sâ€”4558s,1985. massivesecondroundsuperinfectionbymembersofsomesubgroupsofavian 52. elements, J. E., Pedersen, F. S., Narayan, 0., and Haseltlne,W. A. Genomic leukosisvirus.J. Virol.,33:494-506,1980. changesassociatedwithantigenicvariationofVisnavirusduringpersistent 46. Cements, J. E., and Narayan, 0. A physical map of the linear unintegrated infection.Proc.Nail.Aced.Sd.USA,77:4454-4458,1980. DNA of Visna virus. Virology, 113: 412â€”415,1981.

CANCERRESEARCHVOL.45 SEPTEMBER1985 45445

Flossie Wong-Staal, Lee Ratner, George Shaw, et al.

Cancer Res 1985;45:4539s-4544s.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/45/9_Supplement/4539s

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/45/9_Supplement/4539s. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.