[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 10—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(16—18)havebeendetermined.Themost etiologicalagentof theacquiredimmunodeficiencysyndrome(4— 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 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1985 American Association for Cancer Research. HTLVMOLECULARBIOLOGY 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 @—@-———‘@ transcriptionofLTR-linkedgenesiselevatedmanyfoldininfected c=* I [ I T@t LOR4J cells,indicatingatrans-actingtranscriptionalactivationphenom p63 p100? p@ p42 enonviaa viralprotein(10, 31).Thereisindirectevidencethat £!!p24 thex-!orproteinisresponsibleforthisenhancingactivity.Based -@@! I onthesestudies,onecanconstructamodelinwhichthex-!or @@ I I I t I I @!@ç.!! 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(25—27)indicate hasrecentlybeenidentifiedastheetiologicalagentof AIDS,a thatp42isat leastinpartcodedbyx-Ior.Sequenceanalysisof diseasecharacterizedbydepletionof the OKT4-positivehelper theBLVgenomealsorevealsthepresenceofanopenreading