Effect of DNA-dependent protein kinase on adeno-associated virus replication

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TheEffectofDNA-DependentProteinKinaseonAdenoAssociatedVirusReplicationYoung-KookChoi1,KevinNash2¤,BarryJ.Byrne3,NicholasMuzyczka2,SihongSong1*1 DepartmentofPharmaceutics,UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmerica, 2 DepartmentofMolecularGeneticsandMicrobiology,Universityof Florida,Gainesville,Florida,UnitedStatesofAmerica, 3 DepartmentofPediatrics,UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmericaAbstractBackground:DNA-dependentproteinkinase(DNA-PK)isaDNArepairenzymeandplaysanimportantroleindetermining themolecularfateoftherAAVgenome.However,theeffectthiscellularenzymeonrAAVDNAreplicationremainselusive.Methodology/PrincipalFindings:Inthepresentstudy,wecharacterizedtherolesofDNA-PKonrecombinantadenoassociatedvirusDNAreplication.InhibitionofDNA-PKbyaDNA-PKinhibitororsiRNAtargetingDNA-PKcssignificantly decreasedreplicationofAAVinMO59Kand293cells.SouthernblotanalysisshowedthatreplicatedrAAVDNAformed head-to-headortail-to-tailjunctions.Thehead-to-tailjunctionwasloworundetectablesuggestingAAV-ITRself-primingis themajormechanismforrAAVDNAreplication.Inaninvitroreplicationassay,anti-Ku80antibodystronglyinhibitedrAAV replication,whileanti-Ku70antibodymoderatelydecreasedrAAVreplication.Similarly,whenKuheterodimer(Ku70/80)was depleted,lessreplicatedrAAVDNAweredetected.Finally,weshowedthatAAV-ITRsdirectlyinteractedwithKuproteins.Conclusion/Significance:Collectively,ourresultsshowedthatthatDNA-PKenhancesrAAVreplicationthroughthe interactionofKuproteinsandAAV-ITRs.Citation: ChoiY-K,NashK,ByrneBJ,MuzyczkaN,SongS(2010)TheEffectofDNA-DependentProteinKinaseonAdeno-AssociatedVirusReplication.PLoS ONE5(12):e15073.doi:10.1371/journal.pone.0015073 Editor: ImmoA.Hansen,NewMexicoStateUniversity,UnitedStatesofAmerica Received July26,2010; Accepted October19,2010; Published December20,2010 Copyright: 2010Choietal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricted use,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: ThisworkwassupportedbyNationalInstitutesofHealthgrants(P01-DK58327,PO1HL59412,PO1HL51811)toS.S.,N.M.andB.B.,andgrantsfrom JuvenileDiabetesResearchFoundation(JDRF)andAlphaOneFoundationtoS.S.Thefundershadnoroleinstudydesign,datacollectionandanalysis,d ecisionto publish,orpreparationofthemanuscript. CompetingInterests: N.M.isaninventorofpatentsheldbytheUniversityofFloridarelatedtorecombinantAAVtechnology.Healsoownsequityinagene therapycompany(AGTC)thatiscommercializingAAVforgenetherapyapplicationsandservesontheirBoardofDirectors.Hereceivesnomonetary compensationfromAGTCoranyothercommercialentity.Thisdoesnotaltertheauthors’adherencetoallthePLoSONEpoliciesonsharingdataandmater ials. *E-mail:shsong@ufl.edu ¤Currentaddress:MolecularPharmacologyandPhysiology,ByrdAlzheimerInstitute,CollegeofMedicine,UniversityofSouthFlorida,Tampa,Flor ida,United StatesofAmericaIntroductionDNA-PKisanuclearserine/threonineproteinkinasethat consistsofa460kDacatalyticsubunit(DNA-PKcs)anda heterodimer(Ku70andKu80).DNA-PKplaysimportantroles inDNArepairandV(D)Jrecombinationthroughnonhomologous endjoining(NHEJ).WhenDNA-PKencountersDNAlesions suchasDNAdoublestrandbreak(DSB)damagebyionizing radiation,Ku70/80bindswithhighaffinitytoDNAends independentoftheirendsequenceorstructure[1,2,3].TheKu heterodimerrecruitsDNA-PKcstoformanactiveDNA-PK holoenzyme.LigaseIV/XRCC4interactswithDNA-PKonDNA ends,whichleadstoNHEJ[4,5].Severalproteinsincluding Mre11/Rad50/Nbs1andArtemisareinvolvedinthisprocess [6,7].ActivityofDNA-PKcsmayberegulatedbyautophosphorylationofDNA-PKcsatsevenputativephosphorylationsites includingThr2609andSer2056[8,9].Cellsoranimalslacking DNA-PKfunctionsaredeficientinaprotectiveresponseto ionizingradiationandvariousradiomimeticagents[10,11].DNAPKisapotentialtargetproteininmanycancertherapeuticssince inhibitorsofDNA-PKcanselectivelysensitizetumorcellsto ionizingradiation.Wortmannin,aninhibitorofPI3-kinase, inhibitsDNA-dependentproteinkinaseandsensitizescellsto ionizingradiation(IR)[12,13].Inaddition,wortmannindirectly bindstothekinasedomainofDNA-PKcsandinhibitsthefunction ofDNA-PKcsnoncompetitively[14].DNA-PKisasensor moleculethatdeterminesthecellularfatesbyregulatingcellular proteinsrelatedwithcellcycles,DNArepair,andapoptosis [9,15,16,17].Paradoxically,theKu70/80complexcanalsoinhibit nonhomologousendjoiningwhenitbindstothetelomere complex,shelterin[18]. Adeno-associatedvirus(AAV)isanonpathogenichuman parvovirusthatcontainsalinearsingle-strandedDNA(ssDNA) genome[19].TheAAVgenomeencodestwolargeopenreading frames, rep and cap ,thatareflankedby145nucleotideinverted terminalrepeats(ITRs).AAVhasaninterestingbiphasiclifecycle, eitherproductiveinfectioninthepresenceofahelpervirus,e.g., adenovirusorherpessimplexvirus(HSV),orlatentinfectioninthe absenceofahelpervirus.TheITRscomprisetheRepbinding elements(RBEandRBE’)andtheterminalresolutionsite( trs )and formaT-shapedhairpinstructurethatservesastheprimerfor minimaloriginofAAVDNAreplicationandforthesitespecific nickingoftheAAVITRatthe trs thatisrequiredforrepairing covalentlyclosedITRsduringAAVreplication[20,21,22,23].The PLoSONE|www.plosone.org1December2010|Volume5|Issue12|e15073

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largeRepproteins(Rep68orRep78)mediateviralDNA replicationand trs nicking[20,24,25,26,27]andregulateAAV geneexpression[28,29,30,31,32,33,34]andpackaging[35,36]. Rep68orRep78alsoplayimportantrolesforsite-specific integrationofwildtypeAAV2intohumanchromosome 19q13.3qter,namedtheAAVS1locus[37,38,39].AAVDNA replicationrequirestheITR,cellularpolymerases,andhelper virus-derivedfactors.Thep5promoterregionthatregulatesrep geneexpressionisalsoinvolvedinareducedRep-dependent replicationandsite-specificintegrationthatoccursintheabsence oftheITRandreliesontheRBEandcryptic trs inthep5 promoter[40]. InadditiontotheRepproteinsandITRs,AAVDNA replicationrequirescellularproteinsandhelpervirus-derived factorsdependingonthehelpervirusused.InthepresenceofAd, invitro replicationassayssuggestthatfourcellularcomplexesare essentialforAAVDNAreplication;thesearepolymerase d proliferatingcellnucelarantigen(PCNA),replicationfactorC (RFC),andminichromosomemaintenancecomplex(MCM) [25,41,42,43].TheAdandcellularsinglestrandedDNAbinding proteins(DBPandRPA)havealsobeenshowntostimulateAAV DNAreplication invitro [43,44][41].Rephasbeenshownto interactwithallofthesecellularandviralproteins[44,45]. IncontrasttoAdhelperinfections,relativelylittleisknown aboutcellularproteinsinvolvedinAAVreplicationduring coinfectionwithHerpessimplexvirus(HSV).Theherpeshelicase primasecomplexandsinglestrandedDNAbindingproteinare essentialforpromotingAAVDNAreplication invivo ataminimal level[44,46,47].However,expressionoftheHSVDBPand helicase/primaseprovideonly10%ofthenormalDNA replicationseenwithwildtypeherpescoinfection[47].This suggeststhatotherherpesgenesprovideessentialfunctionsand someofthesehaverecentlybeenidentified,(ICP0,ICP4,ICP22) [48].TheherpesDNApolymerase,whichappearstoprovide partialhelperfunctionundersomeconditions[46,48],hasalso beenshowntobecompletelydispensableforrAAVproduction [49],suggestingthatacellularpolymerasemaybenecessaryinthe presenceofHerpescoinfection. WehavepreviouslyreportedthatDNA-PKisinvolvedin determiningthemolecularfateoftherAAVgenomeinskeletal muscleandinliver[50,51].However,theeffectofDNA-PKon AAVreplicationremainselusive.Nashetal(44)haverecently shownthatKu70/80complex,whichformsacomplexwithDNAPK,aswellasDNA-PK,bothformacomplexwithRep78during productiveinfectionsinthepresenceofadenovirus.Theyalso showedthattheDNA-PK/Ku70/Ku80complexstimulatesAAV DNAreplication invitro ,althoughitdoesnotappeartobe essential.InthisreportweexaminetheeffectofDNA-PK/Ku70/ 80onAAVDNAreplicationinthepresenceofHSVandAd.In ordertotesttheeffectofDNA-PKonrAAVreplication,wehave employedboth invivo and invitro replicationassays.We demonstratedthatinhibitionofDNA-PKbywortmanninor siRNAresultedindecreaseofrAAVreplicationinMO59Kand 293cellsinthepresenceofHerpesvirusandin293cellsinthe presenceofAdhelperfunctions.Wealsoconfirmedthatdepletion ofKuproteinsleadtoareductionofAAVreplicationinan invitro assayusingAdinfectedextracts.Results DNA-PKinhibitor,wortmanninInhibitsrAAVreplicationInordertotesttheeffectofDNA-PKonrAAVreplication,we firstemployedwortmannin,aninhibitorofDNA-PK[14].DNAPKpositive( + / + )celllines,MO59Kand293cellswereinfected withrAAV2-UF5withorwithoutrecombinantherpeshelpervirus (containingtheAAV2 rep and cap genes)[52].Cellsweretreated withdifferentconcentrationsofwortmanninandtwodaysafter viralco-infection,episomalDNA(HirtDNA)wasisolated. ReplicatedformsofrAAV-UF5DNAincludingdouble-stranded monomer(about3.4kb),double-strandeddimer(about6.8kb) andhighmolecularweightconcatamers(over13kb)were analyzedbySouthernblotanalysisusing32Plabeledvector specific(CMV)probe.AsshowninFigure1,treatmentof wortmannindecreasedtherAAVgenomereplicationindosedependentmannerinbothcelllines.Nosignificantcytotoxicity wasobservedwhenwortmanninconcentrationwasbelow20mM inMO59Kcells,orbelow5mMin293cells.Wenoticedthatthe conversionofsingle-strandedAAVgenometodouble-stranded monomerorconcatamerswasmoreefficientin293cellsthanin MO59Kcells,probablyduetoE1geneexpressionin293cells [53,54,55].TargetingDNA-PKcsmRNAinhibitsrAAVreplicationTheresultsaboveindicatedthatDNA-PKmayplayarolein enhancingrAAVreplication.However,wortmannininhibitsnot onlyDNA-PK,butalsootherproteinkinases,suchasphosphatidylinositol3-kinase(PI-3).InordertopinpointtheroleofDNAPK,weemployedsyntheticsiRNAtotargetDNA-PKcsmRNA. SyntheticsiRNAat100pmolesignificantlydecreasedmRNA Figure1.rAAVreplicationinMO59Kcellsand293celltreated withwortmannin. HirtDNAwaspurifiedtwodaysafterviralinfection andsubjectedtoSouthernblotanalysis.Allsamplesweretriplicated andhybridizedwith32PlabeledCMVprobe.ReplicatedformsofrAAV includedouble-strandedmonomer(about3.4kb),ordimer(about 6.8kb),andconcatamersDNA(highmolecularweight).Noticethat treatmentofwortmanninreducedrAAVreplicationinadosedependentmannerinbothMO59Kcells(A)andin293cells(B). doi:10.1371/journal.pone.0015073.g001 DNA-PKandAAVReplication PLoSONE|www.plosone.org2December2010|Volume5|Issue12|e15073

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(Figure2A)andproteinlevels(Figure2B)in293cells,while controlsiRNAorLipofectamineTM2000reagentaloneshowedno effect.TheseresultsdemonstratedthatsiRNAwasefficientfor inhibitionofDNA-PKcs.AsshowninFigure3AandB,inhibition ofDNA-PKcsbysiRNAresultedinasignificantdecreaseofrAAV replicationascomparedwithcontrolsiRNAtreatment.The inhibitoryeffectofthesiRNAonrAAVreplicationwasdose dependent(Figure3C).SincetherAAVgenomeislinearand singlestrandedDNA,theviralinfectionmayaffectDNA-PK activity[56,57,58].Toavoidtheunwantedeffectfromviral transductionofrAAVandrHSV[59],weevaluatedrAAV replicationusingtransfecteddoublestrandedvectorandhelper plasmids.Twenty-fourhoursaftersiRNAtransfection,293cells wereco-transfectedwithpDG,whichsuppliesalloftheAdhelper genes,andpUF5.Twodaysafterthetransfection,HirtDNAwas isolatedanddigestedwith Dpn ItoremoveplasmidDNA.Results fromthisstudyagainshowedthatinhibitionofDNA-PK decreasedrAAVreplication(Figure3D).MolecularanalysisofreplicatedrAAVDNAWeandothershavepreviouslyshownthatinlatentinfections, rAAVDNAformsmainlyhead-to-tail(H-T)junctionsandpersists asanepisomeinliverandmusclecells.Cellularenzymes,suchas DNA-PKplayimportantrolesinthejunctionformationand persistenceofAAVDNA[50,51]andwouldbeexpectedto producecircularrAAVgenomeswithheadtotailjunctions. AlthoughAAVisknowntoreplicatebyahairpinpriming mechanism,arollingcirclemechanismhasbeenproposedduring theestablishmentoflatentinfections[60,61].Unliketheselfprimingmodel,whichgenerateshead-to-head(H-H)ortail-to-tail (T-T)junctions,therollingcirclemodelgenerateshead-to-tail(HT)junctions.InordertoevaluatethestructuresofreplicatedrAAV junctionandtheeffectofDNA-PKonjunctionformation, wedigestedHirtDNAwithuniquerestrictionenzymes, Xba I (1-cutter), Sac I(2-cutter),and Not I(2-cutter)asshowninFigure4A. Allofthepossiblefragmentsgeneratedfromthesedigestsarelisted inFigure4B.Southernblotanalysisusingtwodifferentprobes showedthatallpredictedfragments(H-H,T-T,freeends)were detected,exceptH-Tjunctions(Figure4C).Thelowor undetectablelevelsofheadtotailjunctionssuggestedAAV-ITR self-primingwasstilltheprimarymechanismforAAVDNA replication.InhibitionofDNA-PKdidnotaffectthetypeofAAV junctionformedduringAAVreplication.InvitroAAVreplicationInordertoconfirmtheobservationthatinhibitionofDNA-PK decreasedrAAVreplicationincells,weemployedapreviously developedinvitroreplicationassaytoexploretheroleofDNA-PK andKu70/80[25,43].Inthisassay,thereactioncontainspurified Rep68,dsAAVDNAtemplatewithcovalentlyclosedendsand nuclearextract(NE)fromadenovirus-infectedHeLacells.Since HeLacellsexpresshighlevelsofDNA-PK,wetestedtheeffectof selectiveinhibitionofeachsubunitoftheDNA-PKcomplexby addinganti-DNA-PKcs,anti-Ku70,oranti-Ku80antibodiesto theHeLaNEbeforestartingtheAAVreplicationreaction.As showninFigure5A,additionofanti-Ku80antibodysignificantly inhibitedrAAVreplication(70%, P 0.05 ),whileanti-Ku70 antibodyshowedamoderatedecreaseofrAAVreplication (33%, P=0.074 ).However,wedidnotobserveinhibitionof AAVreplicationusinganti-DNA-PKcsantibodies(Ab-23198–4127orAb-4cocktail).Inordertoruleoutanon-specificeffectofthe antibodiesonAAVreplication,weperformedtheassaywithHeLa nuclearextractsthathadbeendepletedforDNA-PKcsorKu heterodimer.SpecificdepletionofDNA-PKcsbyanti-DNA-PKcs Ab-4CocktailandKuproteinsbyanti-Ku70/80(Ku-Ab3)was observedbyWesternblotting(Figure5C).AsshowninFigure5D and5E,depletionofKu70/80decreasedAAVreplication ( P 0.05 byone-taileddistribution),whiledepletionofDNA-PKcsdidnot.Theresultsfromthese invitro replicationstudies confirmedthepreviousreportthatKu70/80interactswithAAV Repproteininvivo,andthattheycanenhanceinvitroAAVDNA replicationandpartiallysubstituteforMCMcomplex[45].AAV-ITRsinteractwithKuproteinsInordertotestforadirectinteractionbetweentheAAVITR andDNA-PK,webuiltanAAV-ITRbyannealingandligating threesyntheticoligonuceotides.ThisITRwasthenlinkedtoa magneticparticle(Figure6A),andasexpected,theITRinteracted withRep78(Figure6B,rightpanel).Inaddition,usingtheITR coatedmagneticparticles,wesuccessfullypulleddownand isolatedKu70andKu80proteins(Figure6B,leftpanel).To eliminatethepossibilitythatincompleteITRsorsinglestranded DNAinteractedwiththeKuproteins,wetreatedtheITRcoated beadswithexonucleaseIIItoremoveanypartiallyassembled ITRs.AsshowninFigure6C,exonucleaseIIItreatmentdidnot affecttheinteractionbetweentheAAV-ITRandKuproteins.The interactionwasalsofoundtobeafunctionoftheconcentrationof Kuproteins.Finally,whenfreecompetitorAAV-ITRwasadded tothereaction,lessKuproteinwaspulleddown(Figure6Dright) althoughthestreptavidin-coatedbeadsaloneweeklyinteractwith Ku70(Figure6Dleft).TheseresultsdemonstratedthatKu proteinscandirectlybindtohairpinedAAV-ITRs,eveninthe absenceofRepprotein. Figure2.TargetingDNA-PKcsbysiRNA. 293cellsaretransfected withsiRNA(100pmole)targetingDNA-PKcsmRNAandnon-specifically controlsiRNAusingLipofectamineTM2000.Twodaysaftertransfection, cellswereharvested.(A)RT-PCRforthedetectionofDNA-PKcsmRNA. AmplifiedcDNAfragmentswereseparatedona1%agarosegel.(B) WesternblotforthedetectionofDNA-PKcsproteinusingbeta-actin proteinasaninternalcontrol. doi:10.1371/journal.pone.0015073.g002 DNA-PKandAAVReplication PLoSONE|www.plosone.org3December2010|Volume5|Issue12|e15073

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DiscussionAAVreplicationhasbeenintensivelyinvestigatedandthe contributionsofAAVITRsequencesandRepproteinshavebeen reasonablywelldefined[20,21,22,23,25,62,63,64,65,66,67,68]. However,theeffectsofcellularproteinsonAAVreplicationare notcompletelyunderstood.Althoughtheminimumsetofproteins requiredtoreplicateAAVDNAefficiently invitro hasbeen identifiedforbothHerpesandAdinfectedcells [41,42,43,44,46,47,49,69,70,71],ithasrecentlybeenreported thatRepproteininteractswith188cellularproteins[45].Someof theseRep-interactingcellularfactorsplayimportantrolesin cellularDNAreplicationorrepairandarelikelytohavearolein AAVDNAreplicationaswell.Understandingthesemechanism(s) willenableustoenhancerAAVvectorproductionandtodevelop asafegenedeliverysystem.Inthepresentstudy,wefocusedonthe roleoftheDNA-PKcomplex(DNA-PKcsandKu70/80)inAAV DNAreplication. OurresultsshowedthatreductionofDNA-PKattheproteinor RNAlevelsdecreasedrAAVreplication,suggestingthatoneor morecomponentsoftheDNA-PKcomplexcanenhanceAAV replication. Invivo ,long-terminhibitionofDNA-PKcsby wortmanninandsiRNAreducedrAAVreplicationinboth MO59Kand293cells(Figs.1,2and3),indicatingthatDNAPKcsplaysanimportantroleinrAAVreplication.Furthermore, DNA-PKcssiRNAreducedAAVDNAreplicationwheneither HerpesvirusorAdhelperfunctionswereused(Fig.3).However, whencell-freeextractswereusedinan invitro replicationassay, depletionofDNA-PKcsdidnotaffectrAAVreplicationsuggesting thattheeffectofDNA-PKcsobserved invivo wasindirectly throughactingondownstreamfactors,suchasKuproteins. Indeed,ourresultsshowedthatKu70andKu80,hadacleareffect invitrowhentheyweredepletedbyeitherantibodyprecipitation orantibodyaddition(Fig.5).Finally,weshowedthatthe componentsoftheDNA-PKcomplex,particularlyKu70/80, couldbindtoahairpinnedAAVITR.Becausethemagnetically taggedsubstrateusedinthesebindingstudieshadaDNAendthat wasblockedwithamagneticbead,ourresultsshowedthedirect interactionbetweenKuproteinsandclose-endedAAT-ITR. Theseresultsareconsistentwithobservationsreportedpreviously usingaChIPassay[72].Ourresultsalsoareconsistentwiththe recentreportbyNashetal[45],whoshowedthatpurifiedKu70/ 80couldpartiallysubstitutefortheMCMhelicasecomplexinan AAVDNAreplicationassay.Thisgroupalsoshowedbyantibody Figure3.TargetingDNA-PKcsreducedrAAVreplication. 293cellsweretransfectedwithDNA-PKcsspecificsiRNA(DNA-PKcssiRNA)orcontrol siRNA.Twodaysaftertransfection,cellswereinfectedwithrAAV-UF5andrHSV,ortransfectedwithpUF5andpDG.rAAVreplicationwasevaluatedby SouthernblotanalysisasdescribedinFigure1.(A)EffectofDNA-PKcssiRNAonrAAVreplicationbySouthernblotaftervectorinfection. ConcentrationofDNA-PKcssiRNAandcontrolsiRNAwas100pmole.(B)DensitometryanalysisofdatafromFigureA.**, P 0.001 formonomer;*, P 0.05 fordimerandconcatamerswhencomparedwithcontrolsiRNAgroup.(C)DosedependenteffectofDNA-PKcssiRNAonrAAVDNA replication.(D)EffectofDNA-PKcssiRNAonrAAVaftervectorandhelperplasmidtransfection.Leftpanel,withoutDpnIdigestion;Rightpanelafte r. Note:DpnIdigestionremovestransfectedplasmidDNAandshowsall denovo replicatedrAAVforms(monomer,dimerandconcatamers). doi:10.1371/journal.pone.0015073.g003 DNA-PKandAAVReplication PLoSONE|www.plosone.org4December2010|Volume5|Issue12|e15073

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co-precipitationthatKu70/80formedacomplexwithRep78and 68 invivo thatwasindependentofthepresenceofDNA.Together, theseresultssuggestthattheenhancingeffectofDNA-PKcsthat weobserved invivo wasprobablythroughphosphorylationofKu proteins,andsuggestedthatKuproteinactivatedbyDNA-Pkcs stimulatedAAVreplication invitro intheabsenceofDNA-PKcs. DNA-PKisaDNArepairenzymeconsistingofalargecatalytic subunit(DNA-PKcs),whichhasser/thrkinase,andaheterodimericcomplexconsistingofKu70andKu80[73].TheKu heterodimerassociatestightlywithdoublestrandedDNAbreaks andrecruitsDNA-PKcs,XRCC4andLigaseIVtorepairthe DNAbreakbynon-homologousendjoining[74,75,76].Kualso bindstotelomeresviaahighaffinityinteractionwithTRF1,a componentofthetelomereshelterincomplexandhasarolein maintainingthestabilityoftelomeres[18].Incontrasttoitsrolein DNArepair,whenKubindstelomeres,itpreventstelomereend joining.Finally,Kuhasalsobeenshowntohavea3 9 to5 9 helicase activityandATPaseactivity[77]andtheseactivitiesappeartobe unrelatedtoitsbindingofDNAends. WeandothershaveshownthatDNA-PKplaysanimportant roleinprocessingrecombinantAAVgenomes[51,72,78, 79,80,81].InjectionofrAAVintoscidmice(DNA-PKcsnegative) showedapersistenceoflinearepisomalrAAVDNAcontaining freeendsinmousetissue,incontrasttonormalmice,inwhichall ofthefreeendshadbeenjoinedpredominantlyinaheadtotail fashion[78,79].Thisresultwasconsistentwiththeprimaryroleof theDNA-PKcomplexinNHEJ,andsuggestedthatthis mechanismpromotedrAAVDNAcirculization,akeystepin establishingstabletransduction.Curiously,Zentilinetal[72] showedthatinshorttermcellcultureexperiments(48hrs), transductionwasincreasedinKu80negativecelllinesinthe presenceofhydroxyurea,suggestingthatalternativemechanisms forformingtransducinggenomescanexistintheabsenceofDNA synthesis.Incontrast,theresultsreportedhereshowedthatAAV DNAsynthesisinthepresenceofhelpervirusfunctionsdidnot resultincircularintermediatesorheadtotailjunctions,regardless ofwhetherKu70/86wasactivatedbyDNA-PKcs(Fig5).This suggeststhattheprimaryroleofKu70/80instimulatingAAV DNAreplicationisunrelatedtoitsroleinpromotingNHEJor transduction.Ourresultsareconsistentwiththerecentreportthat AAVreplicationinthepresenceofAdcoinfectionstimulateda DNAdamageresponsethatwasprimarilyduetoDNA-PKcs[58]. GivenitsinhibitionoftelomereendjoininganditsDNA helicaseactivity,wecansuggesttwogeneralmechanismsbywhich Ku70/80mightstimulateAAVDNAsynthesis.First,the interactionwithRepandtheITRmaypreventNHEJofAAV DNAreplicativeintermediates.Thiswouldbeconsistentwiththe findingthattheproteincomplexcontainingtheAdE4orf6and E1b55KproteinstargetstheMre11/Rad50/Nbs1complex (MRN)fordegradation[82].IntheabsenceofthesetwoAd helperfunctionsforAAV(E1b55KandE4orf6),AdDNAforms concatemers,therebyinhibitingAdDNAreplication.TheMRN complexalsolocalizestoAAVreplicationcentersinthepresence ofAdcoinfectionandinhibitsAAVDNAreplicationinthe absenceoftheE4orf6/E1bcomplex[83].Therefore,thehelper functionprovidedbyE1bandE4orf6isbelievedinparttobethe inhibitionofMRNmediatedNHEJduringAAVDNAreplication. BybindingtoAAVITRs,Ku70/80mayalsopreventinteraction oftheITRwithMRN.Furthermore,bybindingtotheRep-ITR complex,Kuitselfmaybepreventedfromrecruitingtheother Figure4.StructureanalysisofreplicatedDNA. (A)MapoftherAAV-UF5vector;X(XbaI),S(SacI),andN(NotI),Twoboldlinesindicatethe positionofCMVandNeoRprobes.(B)AllpossiblegenomesizesgeneratedfromdifferentAAVjunctions.I.F.,internalfragment;H-H,head-to-head;TT,tail-to-tail;H-T,head-to-tail.(CandD)SouthernhybridizationprobedwithCMV(C),andNeoR(D)afterrestrictionenzymedigestionofHirtDNA. doi:10.1371/journal.pone.0015073.g004 DNA-PKandAAVReplication PLoSONE|www.plosone.org5December2010|Volume5|Issue12|e15073

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componentsoftheDNA-PKNHEJpathway.Thus,bindingofKu totheITRandRepwouldinhibitthetwomajorNHEJpathways inmammaliancells. Ku70/80mayalsoplayaroleinstranddisplacementsynthesis. WehaveshownpreviouslythatAAVDNAreplicationcanbe reconstituted invitro withpurifiedproteins,includingRep,pol d PCNAandRFC[41,42].Hereandelsewhere,wehavealsoshown thatreplicationcanbeatleastpartiallyreconstitutedby substitutingKuforMCM[45].Bothproteinshavesimilar3 9 DNAhelicaseactivitiesthatpreferareplicationforkasasubstrate [77,84];thus,bothcouldfunctioninAAVDNAreplicationas stranddisplacementhelicases.Neitherhasactivityonbluntended DNAmolecules[77,84]and,therefore,theinitialmeltingofAAV endsmaybethefunctionofRep,whichcanbindtoaRepbinding elementwithintheITRandunwindtheend[85].Onceanascent forkisestablished,eitherKuorMCMcanloadontothe3 9 strand andunwindtherestoftheITRtoformthehairpinprimer requiredforloadingDNApolymeraseandexecutingstrand displacementsynthesis. Insummary,wehavepresentedevidenceboth invivo and invitro thattheDNA-PKcomplexandinparticularKu70/80stimulates AAVDNAreplicationinthepresenceofbothAdandHerpes coinfection.Wehavealsosuggestedtwopossiblemechanismsthat mightaccountforthisactivityandguidefutureexperiments.MaterialsandMethods CellsandReagentsAhumangliomacellline(MO59K)wasobtainedfromATCC andwasculturedinDMEM/F-12medium(Cellgrow).Human embryonickidney293cells(Microbix)wereculturedinDulbecco’s MinimalEssentialMedium(DMEM).Allmediaweresupplementedwith10%fetalbovineserum(Cellgro),penicillin(100U/ ml)andstreptomycin(100mg/ml).Wortmannin(Sigma)was dissolvedinDMSOat10mMandstoredat 2 80 u C.rAAVtransductionTherAAV2-UF5vectorwasproducedattheUniversityof FloridaGeneTherapyCenterasdescribedpreviously[86].This vectorcontainsgreenfluorescentprotein(GFP)cDNAdrivenby cytomegalovirus(CMV)promoter.ThetiteroftherAAV-UF5 usedinthisstudywas2.5 6 1013physicalparticles/ml(1 6 1012infectiousunit/ml).TotestAAVreplicationfromviralDNA,cells wereinfectedwithrAAV2-UF5at1000particles/cellorcoinfectedwithoutrecombinantHSVhelpervectorwhichcontains AAV rep and cap genes[52].TotestAAVreplicationfromplasmid DNA,cellsweretransfected1.2mgofhelperplasmid(pDG),and 0.8mgofpTR-UF5byusingLipofectamine2000TM(Invitrogen). HirtDNAwaspurifiedtwodaysafterviralinfectionandwasused Figure5.InvitroAAVreplication. (A) Invitro AAVreplicationusingnuclearextract(NE)supplementedwithanti-DNA-PKcsAb-23198–4127(2,*), anti-DNA-PKcsAb-4cocktail(3,**),anti-Ku80(4),anti-Ku70(5),orwithallantibodies(6).(B)DensitometryanalysisofresultfromFigure5A.Supplement ofanti-ku80decreasedAAVreplicationby30%(DpnI-resistantAAVDNA, P 0.05 comparedtoshamcontrol).(C)Westernanalysisshowsthe depletionofDNA-PKcs,andKu70/80.(D)InvitroAAVreplicationusingDNA-PKcs( D -DNA-PKcs)orKu70/80( D -Ku80/70)depletedNE.Relativedensity ofeachtreatment(n=3)isplotted.*P 0.05. doi:10.1371/journal.pone.0015073.g005 DNA-PKandAAVReplication PLoSONE|www.plosone.org6December2010|Volume5|Issue12|e15073

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forSouthernblotanalysistodetectreplicatedformsofAAV genome.ThedensitometicquantificationofAAVgenomeswas performedwithKodakGelImageSoftware.siRNAtransfectionTwodoublestrandedRNAmoleculeswerepurchasedfrom Ambion(Austin,TX).ThesiRNAforDNA-PKcs(5 9 -GAUCGC ACCUUACUCUGUU-3 9 )targetsthesequencesof352base anddownstreamsequencesofhumanDNA-PKcsmRNA[87]. SilencerTMNegativeControl # 2siRNAfromAmbionwhichdoes notinducenonspecificeffectsongeneexpressionwasusedasa transfectioncontrol.293cellsatthenumberof2.5 6 105cells/well wereculturedin6-wellplatesandtransfectedwith100pmoleof siRNAusingLipofectamineTM2000(Invitrogen).Nextday, siRNAtransfectedcellswereinfectedortransfectedtotestAAV replicationasdescribedabove.RT-PCRTotalRNAwasextractedfromsiRNAtreatedcellsusing TrizolTMreagent(Invitrogen)andRT-PCRwasperformedwith AccessTMRT-PCRkit(Promega)using1mgtotalRNA.Primers werederivedfromthecodingregionofDNA-PKcDNA(upstream primer:5 9 -ACTGACACAGACTGCAGATGGAAG-3 9 downstreamprimer:5 9 -AGGGTGGAAAGAAAGAGAAGG TGG-3 9 ).Beta-actinwasusedasacontrol(upstreamprimer:5 9 TCACCATGGATGATGATATCGCCG-3 9 ,downstream primer:5 9 -ACATGATCTGGGTCATCTTCTCGC). SynthesisofthefirststrandcDNAwasperformedat48 u Cfor 45min.ThePCRwereperformedat96 u Cfor1min,56 u Cfor 1minand72 u Cfor2minfor35cycles.TheamplifiedPCR productswerefractionatedona1%agarosegel.WesternblotanalysisForwesternblotanalysis,cellpelletswerelysedinM-PERTMmammalianproteinextractionreagent(Pierce)supplementedwith proteaseinhibitors(leupeptin,pepstatin,aprotinin,antipain,1mg/ mleach)andPMSF(at1mM).Theproteinconcentrationwas determinedusingBCAkit(Pierce).Thirtymicrogramsoftotal cellularproteinswereseparatedon8%SDS-polyacrylamidegel andtransferredontoanitrocellulosemembrane(Amersham).The mouseanti-DNA-PKcs(DNA-PKcsAb-41–4127)ormouseantiKu80(KuAb-2610–705)ormouseanti-Ku70(KuAb-4506–541)(all antibodieswerepurchasedfromNeoMarkers)wasusedtodetect DNA-PKcs,orKu80orKu70respectively.HorseradishperoxiFigure6.AAT-ITRinteractswithKuproteins. (A)constructionofAAV-ITRonamagneticparticle.(B)ITRonmagneticbeadinteractswithRep78 andKuproteins.AAV-ITRwasboundtopurifiedproteinsorHeLanuclearextract(NE)atroomtemperatureor37 u Candthensubjectedtowestern blotanalysisusingantibodiestoDNA-PKcs,Ku80andKu70.Rep78areusedasapositivecontrol.(C)T-shapedclosedITRinteractwithKuproteinsin dose-dependentmanner.AAV-ITRsonthebeadweretreatedwithExonucleaseIIIandincubatedwithdifferentamountofHeLaNE(65mg/ml).TheITR bindingproteinsweresubjectedtowesternblotanalysisforDNA-PKcs,Ku80andKu70.(D)Competitionassay(rightpanel).WhenAAV-ITRonbead wasincubatedwithHeLanuclearextract,freeAAV-ITRwasadded(2.5fold)asacompetitor.Streptavidin-coatedmagneticbeadsandthebeadswith AAT-ITR(leftpanel)servedasacontrolandshowedadditionofAAV-ITRincreasedpulldownofKuproteins. doi:10.1371/journal.pone.0015073.g006 DNA-PKandAAVReplication PLoSONE|www.plosone.org7December2010|Volume5|Issue12|e15073

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dase-conjugatedgoatanti-mouseIgGwasthenusedandthesignal wasdetectedusingECL(Amersham).InvitroAAVreplicationassayInordertoinhibitDNA-PKsubunits,25mg(1ml)ofAdinfectedHeLacellnuclearextractwaspreincubatedwith1mlof specificantibodiesagainsteachsubunitsofDNA-PK(each 200mg/ml)for30minat37 u C.TheAAVDNAreplicationassay wasperformedasdescribedpreviously[25].Briefly,preincubated Ad-infectedHeLacellextractwithantibodieswasincubatedina reactionbuffercontaining30mMHEPES(pH7.5),7mM MgCl2,0.5mMDTT,100mMeachdNTP,25mCiof[a-32P] dATP,4mMATP,40mMcreatinephosphate,1mgofcreatine phosphokinase,0.1mgofNEsubstrateDNA,and1to80Uof Rep68baculovirusextractfor4hat37 u C.Afterincubation,the reactionmixturewastreatedwithProteinaseKandextractedwith phenol/chloroform.DNAwasprecipitatedinethanol.TheDNA wasdigestedwith Dpn ItoremovebacterialDNAandwas separatedby0.8%agarosegel.X-rayfilmwasexposedonadried gel.ImmunodepletionsInordertodepleteDNA-PKsubunits,25mgofAd-infected HeLacellextractwasmixedwith30mlofanti-DNA-PKcs(DNAPKcsAb-4cocktail),anti-Ku70/80(KuAb-3)orcontrolantibody (mouseIgG2aAb-1,Neomarkers)andincubatedat4 u Covernight. Eachreactionwasaddedwith50mlofProteinG-agarose (Calbiochem)andincubatedat4 u Covernight.DNA-PKsubunits interactedwiththeagarosebeadswerepelletedbycentrifugation. Supernatantswereharvestedcarefully.Thisprocedurewas repeatedthreetimes.Thesupernatantwasusedforinvitro AAVreplicationstudiesasdescribedaboveafterconfirmthe DNA-PKdepletionbyWesternblotanalysis.ConstructionofsyntheticAAV-ITRAT-shapedAAVITRwasbuiltbyannealingandligating threesyntheticoligos,ITR1(65-mer):5 9 -pGCTCGCTCACTGAGGCCGCCCGGGCAAAG CCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA-3 9 ,ITR2(35-mer):5 9 -pGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA-3 9 ITR3(19-mer):5 9 -pTTGGCCACTCCCTCTCTGCGCGCTC3 9 (Figure6A).ThisITRwasligatedwithbiotinatedoligonucleotides,whichcanbindtostreptavidincoatedmagneticparticles (RocheInc).AssayforinteractionbetweenDNA-PKandAAVITRDNAAAVITRonmagneticbeadwasincubatedwithpurifiedDNAPK(containingDNA-PKcs,Ku80andKu70),HeLanuclear extractorRep78atroomtemperatureor37 u C.Theincubation wasperformedinoneoftwobuffersovernightcontainingeither 25mMHEPES(pH7.5),5mMMgCL2,1mMDTTand1% BSAor20mMHEPES(pH7.6),1mMEDTA,10mMNH4SO4, 1mMDTT,0.2%Tween20and30mMKCL.Afterincubation, themagneticbeadswereheldbyamagnetandwashed3times withthebindingbuffer.AAV-ITRbindingproteinswereeluted out,dialyzedovernightfollowingtheinstructionofthekit,and subjectedtowesternblotanalysis.AuthorContributionsConceivedanddesignedtheexperiments:Y-KCKNNMSS.Performed theexperiments:Y-KCKNSS.Analyzedthedata:Y-KCKNNMSS. 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