Proc.Nati.Acad.Sci.USAVol.82,pp.7919-7923,December1985Biochemistry
`Directmappingofadeno-associatedviruscapsidproteinsBandC:
`ApossibleACGinitiationcodon
`(proteinsequencing/humanparvovirus/translationinitiation)
`S.PATRICIABECERRA*,JAMESA.ROSE*,MEDORAHARDYt,BAHIGEM.BAROUDY*,
`ANDCARLW.ANDERSONt
`*LaboratoryofBiologyofViruses,NationalInstituteofAllergyandInfectiousDiseases,NationalInstitutesofHealth,Bethesda,MD20205;andtDepartment
`ofBiology,BrookhavenNationalLaboratory,Upton,LongIsland,NY11973
`CommunicatedbyMaxineSinger,August2,1985
`ABSTRACTThethreemajorcapsidproteinsofadeno-
`associatedvirustype2(AAV2)virionsaredesignatedA,B,and
`Candhavemolecularsizesof90,72,and60kDa,respectively.
`Theseproteinsarerelated,andgeneticstudieshaveshownthey
`areencodedbyalongopenreadingframelocatedintheright
`halfofthegenome.ThecodingcapacitydistaltothefirstATG
`inthisreadingframeisonly503aminoacids(i.e.,aprotein
`aboutthesizeofproteinC),butanopenframesequencedevoid
`ofATGcodonsextendsupstreamforanadditional184codons.
`AlthoughtheaminoterminusoftheCcapsidproteinis
`blocked,partialaminoacidsequenceanalysesofpeptidesfrom
`Chaveconfirmedthatitisencodedwithintheportionofthe
`readingframedistaltothefirstATGatnucleotide(nt)location
`2810.TheaminoterminusoftheBcapsidproteinisnot
`blocked,anditssequencebeginswithalanine.Thetriplet
`encodingthisalaninelies64codonsupstreamfromtheinitia-
`tionsiteforproteinCandisimmediatelyprecededbythe
`threoninecodon,ACG,atnt2615.ThisACGcodonliesinthe
`mostfavorablesequencecontextforproteinsynthesisinitia-
`tion.AllthreeAAV2capsidproteinsarelabeledinvitrowith
`formyl[35S]methionyl-tRNAf,indicatingthatsynthesisofeach
`proteinisinitiatedindependently.Ourdatasuggestthatthent
`2615ACGcodondirectsthemethionyl-tRNA-dependentiniti-
`ationoftheAAV2Bcapsidprotein.ProteinsBandCmaybe
`synthesizedfromthesamemRNAspeciesandtheirrelative
`abundancecouldbedeterminedbytheefficienciesoftheir
`respectiveinitiationcodons.
`Adeno-associatedviruses(AAV)aredefectiveparvoviruses
`whosereplicationrequireshelperfactorssuppliedbyeither
`acoinfectingadenovirus(Ad)orherpesvirus(1,2).TheAAV
`genome,aplusorminussingleDNAstrandof=--4.7kilobases
`(kb),ispackagedinvirionsthatareconstructedwiththree
`majorcapsidpolypeptides(A,B,andC)(3,4).ForAAVtype
`2(AAV2)theapparentsizesofthesecomponentsare90(A),
`72(B),and60(C)kDa,andtheirproportionsinpurified
`virions("1:1:10)aresimilartothosefoundintotalextracts
`ofAd/AAV-coinfectedcells,anindicationthattheirproduc-
`tionmaybetightlyregulated(5).
`Allthreestructuralproteinsareencodedbytherighthalf
`oftheviralgenome,andtheyarespecifiedbyatleasttwo
`mRNAspeciesgeneratedfromtranscriptsinitiatedbythe
`mostrightwardpromoter(mapcoordinate39;Fig.1)(9).
`AlthoughitwasinitiallythoughtthatproteinsBandCwere
`producedbyproteolyticcleavageofproteinA(5),itisnow
`clearthatthesynthesisofBandCdoesnotdependonthe
`synthesisofA(9).Thisfinding,coupledwiththeobservation
`thatproteinsA,B,andCshareaminoacidsequencesthat
`correspondtoallofC(10),suggeststhatthe threestructural
`40 50 60 70 80 90 100
`404148 95
`(1907)(2227)
`i;2.3kbRNA
`2.6kbRNA
`ATG
`(1919)
`DNA
`ATGATGATG
`(1967)(2213)(2810)
`I
`tA5a-1:777
`)3aa="60kdprotein"(C)
`5'RNAU.terminus
`TGATGATAA
`(2165)(2177)(2258)
`3'RNAterminus(4447)
`TAA
`(4322)
`FIG.1.Diagramshowingthelongestright-sidedATG-initiated
`openreadingframeintheAAV2genome.Thereportedunsplicedand
`splicedRNAspeciestranscribedfromtherighthalfofthegenome(6,7)areshownatthetopofthediagram,andthemajoropenreading
`frame(originatingatnt2810)ispositionedontheDNAplus(+)
`strandbelow.Specificgenomicsitesareidentifiedbyntnumber(inparentheses;seeref.8);theindicatedtripletsarein-framewiththe
`ATGthatmapstont2810.ThereisnoATGtripletinanyframe
`betweenthesplicejunctionandtheATGatnt2810.
`proteinsmayoriginatefromindependentin-frameinitiation
`sites.Furthermore,staphylococcalV8proteolysis(10)to-
`getherwithDNAsequencedata(8)indicatethatproteinC
`shouldoccupythelongestright-sidedATG-initiatedopen
`readingframe(mapunits60-92;Fig.1)andshouldtherefore
`overlapequivalentcarboxyl-terminalsegmentsofproteinsA
`andB.Thisreadingframeissufficienttoaccountforprotein
`C,butitisnotlongenoughtoaccommodateproteinswiththe
`apparentsizeofeitherAorB.Thus,theinitiationsitesand
`proximalcodingsequencesfortheAandBproteinsarenot
`evidentfromtheDNAsequenceperse.Janiketal.(9)have
`found,however,thatproteinAisinitiatedfromasitewithin
`theinterveningsequence,whichisintactinthe2.6-kbRNA
`species(Fig.1).TheyproposedthatproteinAmightarise
`eitherbyaread-throughofuptothreeterminationsignalsor
`bytranslationfromahithertoundetectedsplicedRNA
`speciesfromwhichthedownstreamterminatorshadbeen
`ablated(Fig.1).Ontheotherhand,intheabsenceofan
`obviousAUGinitiatorforproteinB,thepossibilitythatB
`Abbreviations:AAV,adeno-associatedvirus;nt,nucleotide(s);kb,
`kilobase(s).
`7919
`Thepublicationcostsofthisarticleweredefrayedinpartbypagecharge
`payment.Thisarticlemustthereforebeherebymarked"advertisement"
`inaccordancewith18U.S.C.§1734solelytoindicatethisfact.
`.Itonl-b--..XIL
`k
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`7920Biochemistry:Becerraetal.
`mightbederivedfromalargerprecursorremainedtobe
`excluded.
`Inthisstudy,wehavedirectlymappedtheoriginsofboth
`theBandCcapsidproteinsofAAV2byaligningtheir
`terminalaminoacidsequenceswithcorrespondingcoding
`sequencesoftheviralgenome.Thesuspectedpositioningof
`Ctotheopenreadingframebetweenmapcoordinates60and
`92wasconfirmed,but,unexpectedly,Bappearedtooriginate
`atanin-frameACGcodon195basesupstreamfromtheATG
`initiatorof C.Inaddition,basedonamino-terminallabeling
`withN-formyl[35S]methionyl-tRNAf(f[35S]Met-tRNAf),we
`haveshownthateachofthethreecapsidpolypeptidesis
`separatelyinitiated.
`MATERIALSANDMETHODS
`InVivoandinVitroSynthesisofRadiolabeledAAVCapsid
`Proteins.293-31cellswerecoinfectedwithAd5(50plaque-
`formingunitspercell)andAAV2(100focus-formingunitsper
`cell)(11).At12hrafterinfection,thecellsweresuspended
`for1hrinaminoacid-freemediumsupplementedwith
`dialyzedhorseserum.Proteinswerethenlabeledwith500
`.uCiof[35S]methionineperml(Amersham;900-1000
`Ci/mmol;1Ci=37GBq)for2hror2.5mCiof[3H]leucine,
`[3H]isoleucine,[3H]alanine,or[3H]prolineperml(Amer-
`sham;56-170Ci/mmol)for3hr.Afterharvesting,nuclear
`extracts,containingthebulkoflabeledAAVcapsidproteins,
`werepreparedasdescribed(9).TolabelAAVstructural
`proteinsinvitro,totalcytoplasmicRNAfromAd2/AAV2-
`coinfectedKBcells(11),isolatedbyCsClcentrifugation(9),
`wasusedtoprogramAAVstructuralproteinsynthesisin
`micrococcalnuclease-treatedrabbitreticulocytelysates.Re-
`actionmixtures(160,u1)contained1mCiof[35S]methionine.
`Topreventacetylationofnascenta-aminotermini,lysates
`werepreincubatedwithcitratesynthaseandoxaloacetateas
`described(12).TopreparefI3YS]Met-tRNAfforamino-
`terminallabelingofAAVcapsidproteins,purifiedyeast
`tRNAfwasaminoacylatedwith[35S]methionine(1440
`Ci/mmol),andformylated[35S]Met-tRNAwasfractionated
`fromnonformylatedtRNAbyRPC-5chromatographyas
`described(13).
`HPLCandAminoAcidSequenceAnalysisofAAVProteins
`andPeptides.AAVcapsidproteins,labeledinvivoorinvitro
`with[Simethionineorwithasingletritiatedaminoacid,
`werepurifiedbyimmunoprecipitationfollowedbyNaDod-
`S04/PAGE(9).Theindividualcapsidpolypeptideswere
`electroelutedandsubjectedtoamino-terminalsequenceanal-
`ysis(14).TrypticpeptideswerefractionatedbyC-18reverse-
`phasechromatographyatpH5.4;individualradiolabeled
`peptideswerepartiallysequencedasdescribed(15).
`RESULTS
`AAVCapsidProteinCMapstotheExpectedOpenReading
`Frame.Eachofthegel-purified[3H]proline-labeledAAV
`capsidproteinsthathadbeensynthesizedinvivowas
`digestedwithtrypsin,andtheresultingpeptideswerere-
`solvedbyHPLCasshowninFig.2.Notunexpectedly,the
`chromatogramsofproteinsA,B,andCshowradioactive
`peakswiththesameelutionpositions(e.g.,P1,P8, P9,and
`P17),whereassomepeaksarenotpresentinallthree
`chromatograms(e.g.,P5intheBandAchromatogramsdoes
`notappearintheCchromatogram).Todeterminethe
`relationshipofproteinCtotheopenreadingframe foundby
`nucleotidesequenceanalysisatmapcoordinate60(Fig.1;
`seeref.8),wedeterminedprolinepositionsintheterminal
`aminoacidsequencesofselectedtrypticpeptidesofprotein
`Candthenmatchedthesetoprolinepositionsintheprotein
`predictedfromDNAsequence.Asanticipated,sequence
`analysesofP1,P8,P9,andP17revealedapatternofproline
`C0
`Q
`Q
`co
`U)0.
`E
`C.)
`a)
`._
`0
`a)
`10
`Fraction(1.0ml)
`FIG.2.Reverse-phaseHPLCoftrypticpeptidesderivedfrom
`AAV2capsidproteins.AAVcapsidproteinslabeledwith[H]-
`prolinewereindividuallypurified,digestedwithtrypsin,andfrac-
`tionatedbyC-18reverse-phasechromatography.Columnswere
`elutedwithadiscontinuouslineargradientofacetonitrile(Lower).
`Theamountofradioactivityineachfractionwasplottedwithout
`backgroundsubtraction.Peaksofradioactivityrepresentingindivid-
`ualpeptidesorpeptidemixtureshavebeenassignednumbersbased
`ontheirorderofelution.Peaksfromeachcapsidcomponentwiththe
`sameelutionpositionwereassignedthesamenumber.Thetotal
`radioactivityappliedtothecolumnwas17,600cpmforcapsid
`componentA(Upper),29,000cpmforcomponentB(Middle),and
`166,000cpmforcomponentC(Lower).Appropriatefractionsfrom
`thechromatogramsofcomponentsBandCwerepooledand
`subjectedtopeptidesequenceanalysis.
`releasethatmatchedpredictedinternalpeptidesofC[nucle-
`otide(nt)locations3545-3580forP1,3500-3544forP8,
`4145-4192forP9,and4058-4117forP17].(Thepositionsof
`thesepeptidesarediagramaticallyshowninFig.SA.)To
`confirmthatproteinCoriginatesfromtheATGatnt2810,we
`triedtoanalyzetheamino-terminalsequenceofintactprotein
`C.SeveralattemptedanalysesofproteinClabeledinvivo
`with[3H]leucine,[3H]isoleucine,or[3H]prolinesuggested
`thatCwasblockedatitsaminoterminus,presumablyby
`acetylationofthepredictedamino-terminalalanineresidue.
`ProteinCwasthereforesynthesizedinvitrounderconditions
`thatpreventedamino-terminalacetylation(seeMaterialsand
`Methods).Amino-terminalanalysesof[35S]methionine-and
`[3H]alanine-labeledproteinCrevealedmethionineinposition
`8andalanineinposition1(datanotshown),resultsconsistent
`withthepredictedoriginofCatnt2810.Inaddition,low
`levelsof[35S]methionineradioactivitywerealsofoundin
`position1,presumablyreflectingtheincompleteremovalin
`vitrooftheinitiatingmethioninefromasmallproportionof
`molecules.WeconcludethatproteinCisencodedbythe
`openreadingframebeginningatnt2810,andthatafter
`removalofitsfirstmethionine,thefollowingalaninebecomes
`acetylatedinvivo.
`Proc.Natl.Acad.Sci.USA82(1985)
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`Proc.Natl.Acad.Sci.USA82(1985)7921
`ProteinBOriginatesUpstreamfromProteinC.Tolocate
`theinitiationpositionofanopenreadingframeforproteinB,
`weanalyzedtheamino-terminalaminoacidsequenceof
`intactproteinBlabeledinvivowith[3H]prolineor[PH]-
`alanineandHPLCtrypticpeptide(P5)ofB(notpresentinC)
`labeledwith[3H]proline.SequenceanalysesofproteinBare
`showninFig.3.The[3H]prolineproteinBanalysisshows
`thattheyieldofradioactivityinresidue2was56%ofthat
`theoreticallyexpectedfromanunblockedproteincontaining
`41prolineresidues(Fig.3Upper).Moreover,[3H]alanine
`proteinBpreparedinvivoalsogaveahighpercentageyield
`ofradioactivityinonlyresidue1,inagreementwiththeamino
`acidsequenceforproteinB(Fig.3Upper).Thissequence
`IIIi III
`APGKKRPVEHSPVEPDSSS
`600 AAV-B
`400-
`200-
`E0.
`correspondstoaproteinbeinginitiatedatanACGcodon(nt
`2615)upstreamandin-framewithproteinC.Fig.3(Lower)
`showstheanalysisofHPLCtrypticpeptideP5,whichwas
`onlypresentintrypticdigestsofproteinsAandB.The
`matchingaminoacidsequenceforP5beginsatthefifth
`residuewithrespecttotheaminoterminusofB,andthe
`entireP5peptidemappedfromnt2630to2686.Aswasthe
`caseforC,amino-terminalanalysisofinvitro-synthesized
`[35S]methionine-labeledproteinBrevealedasmallpeakof
`radioactivityinresidue1.Presumably,thisalsoresultedfrom
`incompleteremovaloftheinitiatingaminoacid residuein
`vitroandsuggeststhattheputativeACGinitiatorspecifies
`methionineastheinitiatingresidue.
`ToconfirmthepresenceoftheACGcodonatthestartsite
`forproteinB,wesequenced(16,17)a927-base-pairrestric-
`tionfragmentofAAVDNA[TthlllI(nt2912)andEcoPJ(nt
`1985);seeFig.SA]derivedfromanAAV2DNAclone(map
`units3-97)containedinpBR325(pLH1).Thisclonehasbeen
`showntospecifyproteinBintransfectionexperiments(9).
`OurresultsagreewiththepreviousAAVDNAsequence
`determination(8)andcorroboratetheoccurrenceofanACG
`tripletatnt2615.
`TheA,B,andCProteinsArisefromIndividualInitiations.
`Purifiedf[15S]Met-tRNAfwasusedtoincorporatemethionine
`attheamino-terminalpositionofAAVpolypeptidechains
`synthesizedinvitroinarabbitreticulocytelysate.ThistRNA
`speciesinitiatespolypeptidesynthesisbytransferringits
`methioninetotheamino-terminalpositionofthepeptide
`chain(18).Formylationofthemethionineresidueblocks
`cleavagefromnascentpolypeptides(19)andf[35S]Met-
`tRNAfshouldnotbeincorporatedatpositionscorresponding
`tointernalAUGcodons.Theelectrophoreticpatternof
`immunoprecipitatedAAVcapsidproteinssynthesizedin
`vitrowithf[35S]Met-tRNAfisshowninFig.4(lane3).Three
`capsidpolypeptideswereproducedthatcorrespondtotheA,
`B,andCcomponentsofpurifiedvirions(lane4).Thereisalso
`correspondencewiththeA,B,andCproteinslabeledeither
`invitro(lane1)orinvivo(lane2)with[35S]methionine.To
`ruleoutinternalincorporationoff[5S]Met,f[35S]Met-labeled
`A,B,andCcapsidproteinswereseparatelydigestedwith
`staphylococcalV8proteaseandwereelectrophoresedtosee
`ifmorethan1peptidefragmentcontainedradioactivelabel.
`Ineachcase,only1ofatleast10fragmentswasfoundlabeled
`(i.e.,theinitialpeptide;refs.10and20).TheA,B,andC
`proteins,therefore,werelabeledonlybytheirinitialfMet
`residues,andweconcludethateachmustoriginatefroman
`independentinitiationsite.
`1 2 3 4
`-- A
`B
`aR.an _ C
`Residue
`FIG.3.SequenceanalysisofAAVcapsidproteinB.(Upper)
`Amino-terminalanalysisofpurified[3H]proline-labeledAAVB
`proteinsynthesizedinvivo(44,500cpmwereappliedtothese-
`quencer).(Lower)AnalysisoftrypticpeptideP5(360cpmapplied)
`derivedfromtheHPLCchromatograminFig.2(Middle).The
`matchingaminoacidsequence(identifiedbythesingle-lettercode)
`derivedfromtheDNAsequenceofAAVisgivenatthetopofeach
`panel.NotethatpeptideP5beginsatthefifthresiduewithrespectto
`theaminoterminusofcapsidcomponentB.
`FIG.4.ElectrophoresisofAAV2capsidproteinslabeledwith
`ft35S]Met-tRNAf.Lanes:1,immunoprecipitatedAAVcapsidpro-
`teinslabeledwith[35S]methionineinarabbitreticulocytelysate
`programmedwithtotalRNAfromAd/AAV-coinfectedKBcells;2,
`AAVcapsidproteinslabeledwith[35S]methionineandim-
`munoprecipitatedfromanuclearextractofAd/AAV-coinfected293
`cells;3,AAVcapsidproteinslabeledwithf[35S]Met-tRNAfand
`immunoprecipitatedfromaninvitroreactionasinlane1.f[35S]Met
`isonlyincorporatedintotheamino-terminalpositionofnewly
`initiatedpolypeptidechains.Lane4,CsCl-purifiedAAVvirions
`labeledwith[35S]methionine.
`Biochemistry:Becerraetal.
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`7922Biochemistry:Becerraetal.
`DISCUSSION
`Wehavedirectlymappedthelocationsencodingtwoofthe
`threeAAV2capsidproteins.AAVproteinC,asexpected,
`mapstotheright-sidedopenreadingframe.Althoughvirion
`AAVproteinCappearstohaveablockedaminoterminus,
`analysisofinvitrosynthesizedChasshownthatCsynthesis
`initiateswiththeAUGencodedatnt2810(Fig.5).From
`analogywithsimilarinitiationsequences,wesuggestthatthe
`initiatingmethionineisremovedandthenewamino-terminal
`alaninebecomesacetylated(12).
`Themajorright-sidedopenreadingframeendswithaTAA
`terminationcodonatnt4322.Wehavenotanalyzedthe
`carboxyl-terminalsequenceofthecapsidproteins,butwe
`haveidentifiedtwoAAVCpeptides(P17andP9)nearthe
`carboxyl-terminalpositionpredictedbythistermination
`codon(Fig.5).AllthreeAAVcapsidproteinscontainthese
`twopeptides.Ifthecapsidpolypeptidesterminatewiththe
`samesequencespecifiedbythent4322TAAcodon,theAAV
`Cproteinwouldbepredictedtoconsistof503aminoacids
`andtohaveamolecularmass(includingacetylgroup)of
`56,278Da.
`DirectsequencingoftheAAVBproteinhaspositionedits
`amino-terminalalanine-encodingsequenceatnt2618.This
`alaninecodonisinthesamereadingframeastheCprotein,
`andnoterminationcodonsoccurinthisframebetweennt
`2618andthebeginningoftheCcodingsequence;therefore,
`Bispredictedtoconsistof568aminoacids,65ofwhichdo
`notoccurinC,andtohaveasizeof62,792Da.Thecodon
`fortheamino-terminalalanineisdirectlyprecededbythe
`threoninecodon,ACG,ratherthantheexpectedATGcodon.
`A
`2.3kbRNA
`EcoRI(1965)
`e7~~(m615)(310)
`P5
`ProteinC
`ProteinB
`AllthreeAAVcapsidproteinscanbeinitiatedinvitrowith
`fMet-tRNAf;thus,Bdoesnotarisebyproteolyticprocessing
`ofA.Furthermore,fI35S]Met-labeledBproteinmigrates
`identicallytoauthenticvirion-derivedBproteinduring
`NaDodSO4/polyacrylamidegelelectrophoresis(Fig.4),and
`thef[35S]Met-labeledstaphylococcalV8protease-derived
`amino-terminalBpeptideandaninternallylabeledV8
`peptidederivedfrom[35S]methionine-labeledBfromvirions
`alsocomigrate.Theseresultsshowthat,atmost,onlyavery
`fewaminoacidscouldberemovedfromthenascentB
`polypeptidetogeneratethealanineamino-terminusofmature
`B.WeconcludethattheinitiationsiteforBmustresidevery
`closetotheamino-terminalalaninecodon(nt2618)intheB
`proteinmRNA;thissitecouldbetheadjacentnt2615ACG
`codon.
`Inhighereukaryotes,onlyAUGhasbeenfoundtofunction
`asaninitiationcodonforproteinsynthesis.Beforeproposing
`thatthent2615ACGcodonservestoinitiatesynthesisofthe
`AAVBprotein,weconsideredtwoalternativeexplanations.
`Onepossibilityisthatduringtranscription,thent2615codon
`ismisreadasAUGwithalowfrequency.Becauseofthe
`favorablecontextsurroundingthent2615site,theresulting
`AUGwouldbeexpectedtofunctionefficientlyasaninitia-
`tioncodon.Weknowofnoprecedentforpolymerase
`misreadingatafrequencyashighas1-5%,theamount
`requiredtoaccountfortheobservedratioofcapsid
`polypeptides.Nevertheless,rigorousexclusionofthispos-
`sibilityisdifficult.
`ThesecondpossibilityforintroducinganAUGcodon
`adjacenttothent2618alaninecodonismRNAsplicing.Two
`separatestudiesofAAVmRNAspecieshavebeenreported
`(4321)
`TthIII(2912)
`4
`Il
`P8Pi
`I3I
`I I
`P17P9
`l'
`B
`5'RNAterminus 19os,2m
`---tTICBCAe ccArcCBT$CABAC6CISAABCTTCBATCAACTACBCABACABATTBBCTCBABBACACTCTCTCTBAABBAATAOACABTBBTBBABCTCAAACCTBBCCCAC29
`SetCysAleAleIeAspVsIAr,Ar,flySerPh.AspC/oL#eArArlSI.II.SlySerArlThrLeSerL#LyeS.cIf.VAspS.rSlySlyicrSrAssLe#AlelHis
`CACCACCAAABCCCBCABABCBBCATAABBACBACABCABBBBTCTTBTBCTTCCTBBITACAABTACCTCBBACCCTTCAACBBACTCBACAASSBABABCCBSTCAACBABSCABACB2410
`#isIis6/CSerProCl/SerSlyllArlThrrhiAleSlyVelLe#CysPh.LeeSlyThrSerrhrSetAspProSefThrAspSe.rhrAr,SGeSrAr,Se.ThrAr,S1Thr
`CCICIICCCTCIABCACTACAAABCCTACBACCBSCABCTCBACABCISASACAACCCITACCTCAASTACAACCACICCSACICSSAlITTCABlABCICCTTAAABAABATACSTCT263
`PieArlProSe.S.,ThrTyrLyeAlaTyrAspArl6/lLeeAsp5crSlyAspAssProTyrLe#LyeTyrAssHisAlaAspAli5/lPh.Cl/G/#Ar,LeeLysSI#AspThrSet
`IB_TTTOffBICAACCTCBOACIAICAITCTYCCABCcAAAABAlITTCTTBAACCTCIBSICCTBITTSA6BAACCITTAA eICC6SAAAALABABSCI6TSAlCACTCT2650Ph*flySlyAseLeeGlyArlAleVolPh.6S/AleLysLyeAr,Vs1LeeCSIPreLee6/7LeeVelSICSI.PreVelLyerTrAlePreGlyLysLysAr,ProVelSI.NisSer
`CCTTlBCCAeACTCCeCCCBAACCBBAAASICScBCCABCABCCTSCAABAAAAABATTCAATTTTBITCABACTISASACBCASACTCA6ThCCTSACCCCCABCCTCTCISA2770
`ProVelC/lPr.Asp5cr5crSerflyrhrSlyLyeAlSlySI#SicProAleAr,LysAr,LeeAssPh.GlySsThrSlyAspAleAspSerVelProAspProC6/ProLeeSly
`_ C
`CABCCACCAICACCCCCTCTBITCTIAACTALTACTIICTACAICAeTSICICACCAA CASACAATAACSAGBBC6CCSACS--- 23Si.Pr.Pr. AleAlePr#5erSrlyLeeSlyTrtAssLrk,ItIAlerhrSlySerClyAlePro1 AleAspAssAss$I.SlyAleAspCly
`FIG.5.(A)SchematicrepresentationofcodingregionsforAAVcapsidproteinsBandCasdeducedfromaminoacidsequenceanalysis.
`Positionsofinternaltrypticpeptidesareshown(hatchedboxes).TheDNAsequencebetweentheEcoRIandTthlllIsites(nt2598-2635)was
`repeatedtoconfirmtheputativeACGinitiationcodonforB.ThesmallestandmostabundantAAVRNAspecies(spliced2.3-kbRNA)is
`representedandmayberesponsibleforthesynthesisofproteinsBandC.(B)DNAplusstrandsequencesthatencodethe5'halfof2.3-kbAAV
`RNA.Thesplicejunctionisidentifiedwithanasterisk.Theindicatedaminoacidsequenceliesin-framewiththemajoropenreadingframe
`initiatedatnt2810.Aterminationsignal(TAA)andmethioninecodons(ATG)areenclosedinsquares.ACGtriplets(threoninecodons)are
`underlinedwithbars.InitiationcodonsforproteinsBandCareindicatedwitharrows.IncontrasttotheotherupstreamACGtriplets,the
`presumptiveBinitiatorliesinthemostfavorablesequencecontextforafunctionalinitiationcodon(21).
`Proc.Natl.Acad.Sci.USA82(1985)
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`Proc.Natl.Acad.Sci.USA82(1985)7923
`(6,22).Bothfoundonlytwotranscripts(of2.3and2.6kb)
`correspondingtothecapsidregion(Fig.1).Webelievethat
`athirdmRNA,splicedatornearnt2615toprovidean
`in-frameAUGcodon,isunlikelytohavebeenmissedifit
`wereatanabundanceinAAV-infectedcellsaS%with
`respecttothe2.3-kbmRNA.
`FurtherargumentsagainstanovelsplicedmRNArespon-
`sibleforBproteinsynthesisarederivedfromananalysisof
`thenucleotidesequencesaroundnt2615andatupstream
`sites(Fig.5).Acompatiblespliceacceptorsitedownstream
`fromnt2620isunlikelybecausenoupstreamATGcodonsare
`followedbyasequencethatcouldencodetheamino-terminal
`sequenceoftheBprotein.Aspliceacceptorupstreamfrom
`nt2600isunlikelybecausetheresultingBproteinprecursor
`(oritsV8proteolyticamino-terminalpeptide)wouldbe
`expectedtomigrateslowerthantheauthenticBequivalent.
`ElevenATGcodonsarefoundbetweenthe5'endofthe
`2.6-kbRNA(atnt1854)andnt2618,butallareintheintron
`(betweennt1907and2227)thatisremovedfromthe2.3-kb
`message.Althoughmanyexamplesofalternativesplicesite
`usagehavebeenreported,wearenotawareofanexample
`wherealternativesplicedmRNAsareproducedsimulta-
`neouslyfromthesametranscriptbyusingbothdifferent
`donoranddifferentacceptorsites.Finally,whilesplicedonor
`andacceptorsitesdisplayconsiderablesequencevariability,
`onerulehasemerged.Intronsalwaysbeginwiththe
`dinucleotidesequenceG-Tand endwithA-G(23).The
`sequenceA-Goccurstwiceimmediatelyupstreamfromnt
`2615(atnt2601-2602andnt2613-2614).NeitherA-Gliesin
`astrikingspliceacceptorcontext[(Y)11NYAG:G].Aspliceat
`eithersitewouldmaintaintheframeofB,andthusnormal
`initiationwouldrequireanin-frameAUGpriortothedonor
`site(nolikelycandidatedonorhasbeenfound);aspliceto
`eithersitewouldrequireadditionalamino-terminalprocess-
`ingtoproducethealanineaminoterminusofB.
`Giventheaboveconsiderations,webelievethemostlikely
`explanationconsistentwithouramino-terminalsequenceof
`Bisthatinitiationofproteinsynthesisoccursatthent2615
`ACGcodon.Asnotedabove,ACGhasnotbeenreportedto
`functionasaninitiationcodonforproteinsynthesisin
`eukaryoticcells;however,theACGtripletdoespromote
`(weakly)thebindingoffMet-tRNAftoEscherichiacoli
`ribosomes(24)andACG,inasyntheticmessage,wasfound
`topromoteinitiationofproteinsynthesis(byE.coli
`ribosomes)invitro(25).
`IfACGservesasaninitiationcodon,thisactivitymay
`requireitsplacementinthemostfavorableinitiationcontext
`(ANNXXXG;seeref.21).In-frameACGtripletsoccuratsix
`positionsbetweentheterminationcodonatnt2258andtheB
`startsite(nt2615;Fig.5),andthreeACGtripletsalsooccur
`inotherreadingframes.Ofthese,nonehasthemostfavored
`context,althoughone,atnt2321,isinthefrequentlyfound
`context:AGGACGACA.AnACGtripletinasimilarcontext
`(AATACGATG)alsooccursimmediatelypriortotheinitia-
`tionsitefortheCprotein.Wehavenoevidencethatanyof
`theseACGtripletspromoteinitiation.Itisperhapsrelevant
`thatararethalassemiahasbeenfoundtoresultfroma
`mutationthathasconvertedtheATGinitiationcodonfora
`humana-2-globingenetoACG(26).ThisACGcodon,like
`theputativeAAVBproteininitiatorliesinthemostfavorable
`initiationcontext,afeaturethatsuggeststhemutationmight
`nothaveentirelyabolishedexpressionofthea-2-globingene.
`ThethreeAAVcapsidpolypeptidesaresynthesizedin
`aboutthesameproportionsasarefoundinvirions.Ifthese
`proportionsarerequiredtoenhanceassemblyoffunctional
`virions,amechanismmustexistforregulatingtheirrelative
`ratesofsynthesis.ThepossibleuseofACGastheinitiation
`codonforproteinBsuggestsaninterestingmechanismthat
`couldregulatetherelativesynthesisofproteinsBandCin
`vivo.IftheseproteinsaretranslatedfromthesamemRNA
`species(i.e.,thespliced2.3-kbRNA;Figs.1and5;ref.9),
`theinitiationcomplexshouldfirstencountertheputative
`ACGinitiatorforproteinB.Assumingthisinitiationsignalis
`relativelyweak,owingtothepresenceofcytosineinsteadof
`uracil,onlyoccasionalinitiationsmightensue(inperhaps
`5-10%ofinstances),withthecomplexusuallymovingonto
`thestrongerAUGinitiatorforproteinC.TheB/Cratio
`wouldthusbefixedandindependentofmessageconcentra-
`tion.Theuseofaninefficientinitiationcodoninthismanner
`mighthavefurtherrelevanceinothereukaryoticsystems.
`WethankDrs.Wei-MayChingandArthurWittwerforproviding
`theE.coliaminoacyltRNAsynthetase,Dr.U.L.Rajbhandaryfor
`purifiedyeasttRNAf,andDr.DolfHatfieldforhisassistancein
`purifyingf[35S]Met-tRNAf.WealsothankDr.EdwinSebringand
`Mr.KeithThompsonforcomputeranalysesandJoyceGloverfor
`typingthemanuscript.ThisworkwassupportedinpartbytheUnited
`StatesDepartmentofEnergy.
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`Sarepta Exhibit 1011, page 5
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