The Open Reading Frame VI Product of Cauliflower mosaic virus Is a Nucleocytoplasmic Protei

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

ThePlantCell,Vol.17,927–943,March2005,ª2005AmericanSocietyofPlantBiologists

TheOpenReadingFrameVIProductofCauli owermosaicvirusIsaNucleocytoplasmicProtein:ItsNTerminusMediatesItsNuclearExportandFormationofElectron-DenseViroplasms

`leGeldreich,aMarinaBureau,aLaurenceDupuis,aVe´roniqueLeh,a,2GuillaumeVetter,aMurielHaas,a,1Ange

KappeiKobayashi,bThomasHohn,bLyubovRyabova,aPierreYot,aandMarioKellera,3

´culairedesPlantes,Unite´PropredeRecherche,CentreNationaldelaRecherchedeBiologieMole

´LouisPasteur,67084StrasbourgCedex,FranceScienti que2357,Universite

bFriedrichMiescherInstitute,CH-4002Basel,Switzerland

aInstitut

TheCauli owermosaicvirus(CaMV)openreadingframeVIproduct(P6)isessentialfortheviralinfectioncycle.ItcontrolstranslationreinitiationoftheviralpolycistronicRNAsandformscytoplasmicinclusionbodies(viroplasms)wherevirusreplicationandassemblyoccur.Inthisstudy,themechanisminvolvedinviroplasmformationwasinvestigatedbyinvitroandinvivoexperiments.FarproteingelblotassaysusingacollectionofP6deletionmutantsdemonstratedthattheN-terminala-helixofP6mediatesinteractionbetweenP6molecules.Transientexpressionintobacco(Nicotianatabacum)BY-2cellsoffull-lengthP6andP6mutantsfusedtoenhancedgreen uorescentproteinrevealedthatviroplasmsareformedattheperipheryofthenucleusandthattheN-terminaldomainofP6isanimportantdeterminantinthisprocess.Finally,thisstudyledtotheunexpected ndingthatP6isanucleocytoplasmicshuttleproteinandthatitsnuclearexportismediatedbyaLeu-richsequencethatispartofthea-helixdomainimplicatedinviroplasmformation.ThediscoverythatP6canlocalizetothenucleusopensnewprospectsforunderstandingyetunknownrolesofthisviralproteininthecourseoftheCaMVinfectioncycle.

INTRODUCTION

Cauli owermosaicvirus(CaMV)isthetypememberoftheCaulimovirusgenusoftheCaulimoviridaefamily.Itsdouble-strandedcircularDNA(;8kb)isreplicatedviaanRNAin-termediatebyavirus-encodedreversetranscriptase,andthevirusisclassi edinthepararetrovirussupergroup,towhichanimalvirusesoftheHepadnaviridaefamilyalsobelong(forareview,seeHaasetal.,2002).TheCaMVgenomepossessessixmajoropenreadingframes(ORFsItoVI),whicharealllocatedonthesameDNAstrand.ManyfunctionsofthecorrespondinggeneproductsP1toP6havebeenelucidated,butthemecha-

´PropredeRecherche,CentreNationaldelaaddress:Unite

´partementRe´cepteursetProte´inesRechercheScienti que9050,De

´rieuredeBiotechnologiedeStrasbourg,Membranaires,EcoleSupe

´bastientBrant,67412IllkirchCedex,France.boulevardSe

2Currentaddress:LaboratoiredeDynamique,EvolutionetExpression

´nomesdeMicroorganismes,FormationdeRechercheendesGe

´Evolution2326,CentreNationaldelaRechercheScienti que,Universite

LouisPasteur,28rueGoethe,67084StrasbourgCedex,France.

3Towhomcorrespondenceshouldbeaddressed.E-mailmario.keller@ibmp-ulp.u-strasbg.fr;fax33-3-88-61-44-42.

Theauthorresponsiblefordistributionofmaterialsintegraltothe ndingspresentedinthisarticleinaccordancewiththepolicydescribedintheInstructionsforAuthors()is:MarioKeller(mario.keller@ibmp-ulp.u-strasbg.fr).versioncontainsWeb-onlydata.

Article,publicationdate,/cgi/doi/10.1105/tpc.104.029017.

1Current

nismsbywhichtheyoperateduringviralinfectionarenotyetfullyunderstood.

TheviralDNAistranscribedbythecellularRNApolymeraseIIintotwomajorcappedandpolyadenylatedRNAs,amonocis-tronic19SmRNAandapregenomic35SRNAthatservesasatemplatebothforreversetranscriptionandfortranslationintoP1toP5.The35SRNAundergoesalternativesplicingeventsleadingtofourmRNAsinwhichORFIandpartofORFIIaredeleted(Kiss-Laszloetal.,1995).Currently,themechanismregulatingthenuclearexportof35SRNAanditssplicedformsisunknown.TheseRNAsaretranslatedbythecellularmachineryfollowingtwounconventionalstrategies,ribosomalshuntandtermination–reinitiation(forareview,seeRyabovaetal.,2002).TheCaMVP6protein(62kD),whichisexpressedspeci callyfromthe19SRNA,isamultifunctionalproteinthatrepresentsakeycomponentintheCaMVinfectiouscycle.P6isthemajordeterminantofhostspeci cityandin uencessymptomseverity(Daubertetal.,1984;DaubertandRouth,1990;Agamaetal.,2002).InoculationofcruciferousandsolanaceousplantspecieswithchimericCaMVgenomesbearingORFVIderivedfromdifferentCaMVisolatesshowedthattheN-terminalregionofP6isresponsibleforhostspeci city(Daubertetal.,1984;Schoelzetal.,1986).TransgenicArabidopsisthalianaplantsexpressingP6displaydiseasesymptomswhoseseverityisrelatedtotheexpressionlevelofthetransgene(Zijlstraetal.,1996).Compar-isonofthecellularmRNAcontentofORFVI-transgenicandcontrolArabidopsisplantsrevealedthatORFVIexpressiondownregulatesorupregulatesseveralhostgenes(Gerietal.,

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

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1999).WhetherP6playsadirectroleinregulatingexpressionofthesecellulargenes(i.e.,byregulatingtheirtranscription)hasnotbeendetermined.Finally,P6fromcertainCaMVisolatescanalsoactasanavirulencegeneproducttopromoteahypersensitiveresponseinsomeNicotianaspecies(Palanichelvametal.,2000;Coleetal.,2001).

P6trans-activatestranslationoftheviralpolycistronic35SRNAanditssplicedversionsandhenceallowssynthesisofacompletesetofviralproteins(forareview,seeRyabovaetal.,2002).Parketal.(2001)demonstratedthatP6isatranslationalreinitiationfactorthatassociateswiththehosttranslationalmachineryandthuspermitstranslationofdownstreamORFs.Thisfunc-tionismediatedbyphysicalinteractionsbetweenaninternalregion,includingtheminimalsequenceofP6requiredfortrans-activation(themini-TAV;DeTapiaetal.,1993),theinitiationfactoreIF3(Parketal.,2001),andtheribosomalproteinsL13(Bureauetal.,2004),L18(Lehetal.,2000),andL24(Parketal.,2001).TheobservedinteractionbetweenP6andtheCaMVcapsidprotein(P4)alsosuggestsaroleforP6asascaffoldingproteinintheassemblyofCaMVparticles(Himmelbachetal.,1996).

P6isanabundantlysynthesizedCaMVprotein,andinthecytoplasmofinfectedcellsitformsamorphous,non-membrane-limited,electron-denseinclusionbodies,alsoreferredtoasviroplasms.Theseelectron-denseviroplasmsaredistinctfromtheelectron-lucentinclusionbodiesthataremainlycomposedofCaMVP2protein(Espinozaetal.,1991;Druckeretal.,2002).Thenumberandthesize(2to10mmindiameter)oftheelectron-denseviroplasmsdependonthestageoftheviralcyclebutalsoontheCaMVisolateandthehostplant(Shallaetal.,1980).Electron-denseviroplasmsareahallmarkofinfectionofplantcellsbycaulimovirusesandsoymoviruses.Theyprobablyplayanimportantroleintheinfectiouscyclebecausetheyarethesitesofproteinsynthesis,viralgenomereplication,morphogen-esis,andstorageofthenewlyformedvirions(Mazzolinietal.,1989;Rothnieetal.,1994).OtherCaMVproteinshavebeendetectedintheelectron-denseviroplasms(Druckeretal.,2002),butnoneofthemseemstoberequiredfortheirformationbecausetransgenicArabidopsisplantsexpressingP6alonecontaininclusionbody-likestructures(Cecchinietal.,1997).PreviousdatafromseveralstudiessuggestedthatP6self-associates(Leh,1999;Haasetal.,2000),andLiandLeisner(2002)showedthatmultipledomainswithinP6caninteractwiththefull-lengthprotein;theyproposedthattheseinteractionsmightbeinvolvedintheformationofviroplasms.

Inthisarticle,wedemonstrateforthe rsttimethatP6localizesbothinthecytoplasmandinthenucleusofplantcellsandthattheN-terminalregionofP6hasadualfunction.ItisamajordeterminantforinvitrointeractionbetweenP6moleculesandmediatestheformationofviroplasmsinvivo.ItalsocontainsaLeu-richnuclearexportsignalthatpreventsaccumulationofP6moleculeswithinthenucleus.RESULTS

TheN-TerminalRegionMediatesP6–P6InteractionsinVitroInpreviouslydescribedfarproteingelblotexperiments(Leh,1999),proteinsfromhealthyandCaMV-infectedturnip(Brassicarapa)ingP6expressedinEscherichiacoliand32P-radiolabeledinvitroasoverlay,aradioactivesignalwasdetectedwithproteinsfrominfectedplantsatthelevelofapolypeptideof62kDthatalsoreactedwithanti-P6antibodies,stronglysuggestingthatP6interactswithitself.However,becauseCaMV-P6proteindownregulatesorupregulatestheexpressionofseveralhostproteingenes(Gerietal.,1999),itcouldnotbetotallyexcludedthattheblot-immobilizedspeciesinteractingwith32P-P6intheaboveexperimentwasahostproteinofsimilarmobilitytoP6thatwasexpresseduponviralinfection.Toruleoutthispossibility,wehaveperformedanidenticalfarproteingelblotexperimentexceptthattheimmo-bilizedproteinsontheblotwereobtainedfromanextractofE.coliexpressingP6.The32P-P6intheoverlayagainreactedwitha62-kDspecies(Figure1C,laneP6),whichwasalsorecog-nizedbyanti-P6antibodies(Figure1B,laneP6),providinginde-pendentcon rmationthatP6caninteractwithitself.Asimilarresultwasobtainedusingapull-downassay(datanotshown).BecauseP6containsseveraldomainsthatcanbindsingle-and/ordouble-strandedRNAandRNA-DNAheteroduplexes(DeTapiaetal.,1993;Cerritellietal.,1998),farproteingelblotassayswerealsoperformedaftertreatmentofboththeoverlayandthemembrane-boundproteinswithRNaseandDNase.ThesetreatmentsdidnotimpairformationoftheP6-P6complex,de-monstratingthatneitherRNAnorDNAmediatestheP6–P6interactionandconsequentlythatoneormoredomainsofP6aredirectlyinvolved.

Tocharacterizetheregion(s)requiredforself-associationofP6,wetestedthecapacityofaseriesofP6deletionmutants(Figure1A)tobindfull-lengthP6.ThemutantscorrespondedtoN-andC-terminalrecurrentdeletionsandtoP6bearinginternaldeletionsofpreviouslyidenti edfunctionaldomains(i.e.,themini-TAVandRNAbindingsites)(DeTapiaetal.,1993).ThedeletedproteinswereexpressedinE.colifrompET3arecombi-nantplasmids,separatedbySDS-PAGE,blottedontoanitrocel-lulosemembrane,andincubatedinthepresenceof32P-labeledP6intheoverlaysolution.AsshowninFigure1C,P6interactedwiththeC-terminaldeletionmutantsA,B,C,D,andEandwiththeinternallydeletedmutantsJ,K,L,andM,butnotwiththeN-terminaldeletionmutantsF,G,H,andI.Becausenoradio-activesignalscouldbedetectedwiththelattermutantseventhoughtheywerepresentinrelativelyhighamountsonthemem-brane(Figure1B,lanesFtoI),wecanruleoutthepossibilitythatthesignalsobservedwiththeotherP6mutantscorrespondtoartifactualbindingasaresultofgrossoverloadingofthemembranewithproteins.TheminorsignalsobservedinlanesJtoM(Figure1C)correspondtointeractionsbetweentheoverlayandP6degradationproductsratherthantononspeci cinter-actionswithbacteriaproteinsasdemonstratedbythecontrolexperimentperformedwithbacteriatransformedwithemptyvector(Figure1C,laneE.coli).

ThesmallestmutantabletointeractwithP6wasmutantA,whichcorrespondstothe112N-terminalaminoacidsofP6,aregionwewillrefertoasdomainA.ThelatterwasfusedtotheNterminusofaproteinfromanunrelatedvirus,P42ofBeetnecroticyellowveinvirus,ortotheCterminusofglutathioneS-transferase(GST)toanalyzetheabilityofdomainAtointeract

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

CaMVP6IsaNucleocytoplasmicProtein929

Figure1.MappingoftheP6DomainInvolvedinP6–P6Interactions.

(A)Schematicrepresentationoffull-lengthP6(520aminoacids[aa])andP6deletionmutantsusedinthefarproteingelblotassays.Theminimalsequencerequiredfortranslationaltransactivation(mini-TAV),thesingle-strandedRNAbindingdomains(ssRNA),andthezinc ngermotif(Zn)areindicatedbydarkgray,black,andgrayboxes,respectively.ThemolecularmassesofP6anditsdeletedversionsareindicatedtotheright.

(B)and(C)Bacterialextractscontainingfull-lengthP6(laneP6),truncatedversionsofP6(lanesAtoM)ornot(laneE.coli)wereseparatedbySDS-PAGE(15%)andtransferredontoanitrocellulosemembrane.ThemembraneswereeitherincubatedwithantibodiesraisedagainstP6(B)orsubmittedtoafarproteingelblotassayusinginvitro32P-labeledP6(C).

(D)The112N-terminalaminoacidsofP6correspondingtomutantAwerefusedtoanunrelatedprotein,P42ofBeetnecroticyellowveinvirus.ThefusionproteinwasexpressedinE.coli,separatedbySDS-PAGE,andtransferredontoanitrocellulosemembrane.Themembranesweresubmittedtoafarproteingelblotassayusing32P-labeledP6asaprobe(leftpanel)ortoproteingelblottingusingspeci cpolyclonalantibodies(rightpanels).Molecularmassesofmarkerproteinsusedintheexperimentsareindicatedattheleft.

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

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withP6whenplacedinanunrelatedsequencecontext.P6boundtoA:P42butnottoP42alone(Figure1D)andtoGST:AbutnottoGST(seebelow),thusfurtherdemonstratingthatdomainAcanactindependentlyoftherestoftheaminoacidsequenceinmediatingP6self-associationinvitro.Takentogether,theresultsoftheabovefarproteingelblotexperimentsprovideevidencethattheN-terminalregionencompassesthedomainrequiredforP6self-interactioninvitro.

TheNTerminusofP6IsanEssentialDeterminantforBoththeFormationofViroplasmsandTheirLocalizationintheCytoplasm

ToobtainfurtherinformationabouttheimportanceoftheN-terminalregionofP6intheformationofinclusionbodies,tobacco(Nicotianatabacum)BY-2cellsweretransfectedwithrecombinantpCK-EGFPplasmidscodingforfull-lengthP6andtwodeletedversions(AandP6DA)fusedtotheCterminusoftheenhancedgreen uorescentprotein(EGFP).Theresultsde-scribedbelowarerepresentativeofatleastfourindependenttransfectionexperimentsandobservationbyconfocallaserscanningmicroscopy(CLSM)at20hpost-transfection.

AfterbombardmentofBY-2cellswithplasmidsexpressingtheEGFP:P6fusionprotein,;80%oftransfectedcellscontainedlargecytoplasmicinclusionbodies(3to5mmindiameter)withpittedsurfaces,generallyintheproximityofthenucleus(Figure2A,panels1and2).Theinclusionbodieswereformedbynumeroussmalleraggregates,mostofwhichappearedashollowdonut-likestructures(Figure2A,panel3).Toexcludethepossibilitythattheirformationwasartifactual(i.e.,asaresultoftheEGFPmoietyfusedtotheP6),protoplastswerepreparedfromCaMV-infectedturnipplantsasdescribedbyKobayashietal.(1998), xedandimmunolabeledwithanti-P6andsecond-aryantibodiescoupledtothe uorochromeAlexa568.Obser-vationsbyCLSMrevealedthattheviroplasmsthusproducedinthecontextofanauthenticviralinfectionhadasimilarstructure(Figure2B),demonstratingthattheEGFPmoietyhasnopro-nouncedeffectontheself-assemblyofEGFP:P6intobaccocells.MoreovertheseresultsalsoillustratethatP6moleculesassembleproperlyandindependentlyofthecellularcontextbecausesimilarlyshapedaggregateswereformedincellsfromhost(turnip)andnon-host(tobacco)plants.

Approximately20%ofthetobaccocellsexpressingtheEGFP:P6fusionproteincontainedaggregatesofvariablesizesbut<2mmindiameter(Figure2A,panels4and5),whichprobablycorrespondtoearlystagesofviroplasmformation.Thesmalleraggregatesgenerallywerescatteredinthecyto-plasm,althoughtheywerealsosometimesfoundwithinthenucleuswhenthecellswereanalyzedbyCLSM.ThepresenceofsuchaggregateswithinthenucleusmayindicatethatEGFP:P6moleculesweretransportedtothenucleus(seebelow)andwerethenunabletoexitaftertheirself-assemblybecauseofthelargesizeoftheresultingaggregates.

IncontrastwithEGFP:P6,EGFP:P6DA(Figure3A)didnotformaggregatesintobaccocells(Figure3B,panels1and2)butwasmainlyfoundinthenucleusandinparticularwithinthenucleolus.ThisresultstronglysuggeststhattheN-terminalregionofP6isadeterminantnecessaryfortheformationofaggregatesandthat

italsocontainssignal(s)involvedinthetargetingand/orretentionofP6withinthecytoplasm.SimilarlytoEGFP:P6DA,expressionofEGFP:Anevergaverisetoaggregatesofanysize,buttheproteinwasinsteaddistributeduniformlyinthecytoplasm(Figure3B,panels3and4).ThefailureofEGFP:Atoaccumulateinthenucleustoasigni cantextentwassomewhatsurprisingbecauseitissuf cientlysmallthatitwouldbeexpectedtobeabletotraversenuclearporesbypassivediffusion.

Takentogether,thesedatashowthattheN-terminalregionofP6isnecessarybutapparentlynotsuf cientfortheformationofviroplasmsand,thus,thatotherregion(s)ofP6contributetothisprocess.OurfailuretodetectEGFP:AinthenucleusstronglysupportstheideathatthecytosoliclocalizationofP6isgovernedbydomainA.

TheN-TerminalP6–P6InteractionDomainIsConservedamongCaMVStrains

ComparisonoftheN-terminalsequenceofP6fromCaMVstrainCabb-BJIwithitscounterpartsfromotherCaMVstrains(CabbS,CabbS-Japan,NY8153,CM1841,W260,D/H,D4,B29,Xin/Jin,andBari)showedidentityrangingfrom83to97%.TheregioncanbedividedintotwosubdomainsthatwehavedesignatedA1(aminoacids1to83)andA2(aminoacids84to112),respectively(Figure4A).Theformeristhemostconserved(87to99%ofidentity)withtwonotableinvariantsequences:I1(aminoacids11to20)andI2(aminoacids63to83).NotethatI1alsocontainsapentapeptidemotifEKILM(residues11to15)thatisidenticalatfourof vepositionstotheupsteamsequenceEKLLM(motifi1;Figure4B,top).ThesequenceI1formspartofapredicteda-helixlocatedneartheNterminus(positions4to31)thatcontainsaseriesoffoursuccessiveheptadsequences(abcdefg),whereresiduesinpositionsaanddarehydrophobicasinLeuzippermotifs(Figure4B).Thissequenceispredictedbycomputeranalysis(Berger,1995)todimerizeviaacoiled-coilstructurewith>98%probability.SubdomainA2ismorevariable(62to93%identity)anddoesnotpossessaconservedmotifamongCaMVstrains;ithasapredictedbsheetcon guration.

Asa rststeptowardfurtherde ningtheP6–P6interactiondomain,thesequencescodingforsubdomainsA1andA2wereampli edbyPCRandclonedintothepGEX-2TKvectortoproduceGST-taggedproteins.Thecapacityofthesefusionproteinstoself-interactandtointeractwithP6orGST:Aweretestedbyfarproteingelblotexperiments.RadiolabeledP6interactedwithGST:AandGST:A1butnotwithGST:A2(Figure4C,leftpanel),showingthatsubdomainA1isresponsiblefortheinteractionbetweenP6molecules.Thisresultwascon rmedbythe ndingthat,wheneitherGST:AorGST:A1wasusedasoverlay,theyinteractedwithP6andwiththemselvesbutnotwithGST:A2,whereasthelatterwasunabletobindeithertoP6ortoanyofthefusionproteins(Figure4C,rightpanels).NotethatnoneofthefusionproteinsinteractedwithGST,excludingthepossi-bilitythattheobservedinteractionswereduetodimerizationofthetag.Takentogether,theseresultsprovideevidencethatthe83N-terminalaminoacidsencompassthedomainrequiredforP6self-interactioninvitroand,hence,probablyfortheformationofviroplasms.

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

CaMVP6IsaNucleocytoplasmicProtein931

Figure2.SubcellularLocalizationAnalysisofP6FusedtoEGFPinTobaccoBY-2CellsandofP6inProtoplastsfromCaMV-InfectedTurnipPlantsbyCLSM.

(A)Green uorescentimagesofEGFP:P6(images1and4)weretaken20haftertransfectionoftobaccocellsbybombardmentwithpCK-EGFP:P6plasmid.A0.45-mm-thickopticalsectionwassampledusingasingletrackconfocalmicroscopeandappropriate lters.Image3correspondstoanenlargementofaggregatessimilartothoseobservedinimage1.Images2and5correspondtothesuperpositionofthe uorescentimageandthecorrespondingdifferentialinterferencecontrastimage.Bars¼10mm.N,nucleus;Nu,nucleolus.

(B)ProtoplastspreparedfromCaMV-infectedturnipleaveswere xedandimmunolabeledwithrabbitanti-P6antibodiesandmouseanti-rabbitIgGcoupledtoAlexa568assecondaryantibody.Shownisthered uorescentimageofatypicalprotoplast.Theconfocalimageswerecollectedwithafocaldepthof0.45mm.Bar¼10mm.

MutationsintheN-Terminala-HelixofP6AffecttheFormationofViroplasms

InviewofthefactthatsubdomainA1istotallyconservedamongCaMVstrainsandispartoftheputativeLeuzipper–containinga-helix,additionalexperimentswereperformedtofurtherin-vestigateitsroleinviroplasmformation.Weremovedboththei1andI1sequences(aminoacidsfrompositions5to20)fromP6(Figure5A)toseeifthereductionofthesizeofthea-heliximpairstheformationofviroplasms.The uorescenceofthecorrespond-ingEGFP:P6Di1-I1mutantwasveryabundantanddiffuseinthenucleus,whereasonlylowlevelswerefoundinthecytoplasm(Figure5B,panels1and2).SimilarbehaviorwasobservedwithmutantEGFP:P6DI1,inwhichwedeletedonlytheI1sequence(Figure5B,panels3and4).TheseP6mutantsdidnotformaggregates,thusreinforcingthehypothesisthattheN-terminala-helixisrequiredforP6self-assembly.Moreover,theseresultsstronglysuggestthatthea-helixand/orspeci cresiduesofI1arealsoimplicatedinthecytoplasmiclocalizationofP6becausebothconstructions,EGFP:P6Di1-I1andEGFP:P6DI1,localizedalmosttotallyinthenucleus,incontrastwithEGFP:P6(Fig-ure2A).

Insummary,theforegoingresultsstronglysuggestthattheN-terminala-helixhasstructuralfeaturesimportantforboththeaggregationofP6anditslocalizationinthecytoplasm.Todemonstrateitsroleintheformationofviroplasm,wemutatedthreeLeuresiduesoftheI1sequenceofP6(seealsobelow).TheLeuresiduesatpositions14and16weresubstitutedbyGlnresiduesandtheLeuatposition18byaHis(Figure4B,top).Aminoacidresidues14and18correspondtoposition‘‘d’’ofthesecondheptadandtoposition‘‘a’’inthethirdheptadoftheLeuzipper.Theyarepredictedtolieonthesurfaceofthea-helixandtobeinvolvedinhydrophobicinteractionsbetweenP6mole-culesinthecoiled-coilstructure(Figure4B).Leu16,ontheotherhand,isnotpredictedtobesurface-located.

First,wetestedthecapacityofthismutant,namedP6m1,tointeractwiththewild-typeP6byfarproteingelblotassay.

The

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

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Figure3.SubcellularLocalizationofTruncatedVersionsofEGFP:P6inTobaccoBY-2Cells.

(A)Schematicrepresentationoffull-lengthP6andoftruncatedversionsfusedtotheCterminusofEGFP.P6fragments:thenumberscorrespondtoaminoacidpositionswithintheclonedP6sequence.EGFPisrepresentedbyagreenboxanddeletedversionsofP6byanemptybox,exceptdomainA,whichisyellow(nottoscale).TheotherdomainsofP6arerepresentedasinFigure1.CLSMimages:thenumbersreferto(B).aa,aminoacids.(B)SubcellularlocalizationofEGFP:P6mutants(1to4)intobaccoBY-2cells20hpost-transfectionbybombardment.FluorescenceimageswerecollectedbyCLSMasdescribedinFigure2.Images2and4correspondtothesuperpositionofthe uorescentimageandthecorrespondingdifferentialinterferencecontrastimage.Bars¼10mm.

proteinwasexpressedinE.coli,fractionatedbySDS-PAGE,transferredontoanitrocellulosemembrane,andthenincubatedwith32P-labeledP6.AsshowninFigure6,P6m1nolongerinteractedwithP6,suggestingthattheputativehydrophobicbondsinvolvingLeu14and18arecrucialforformationoftheP6-P6complex(cf.tolaneP6correspondingtothepositivecontrol).WealsofusedP6m1totheCterminusofEGFPandexpresseditinBY-2cells.VisualizationofEGFP:P6m1byCLSMrevealedthatitneverproducedaggregatesbutwasinsteadevenlydistributedthroughoutthecytoplasmandthenucleus(Figure5B,panels5and6)incontrastwithEGFP:P6deletedversionsthatmainlyaccumulatedwithinthenucleus(Figure5B,panels1to4).WealsodeterminedbyAlascanningwhetherotherresiduesofthea-helixareinvolvedintheaggregationofEGFP:P6.InthemutantEGFP:P6m2,theMet,Glu,andAspresidueslocatedbetweenLeuresiduesoftheI1sequenceatpositions15,17,and19,respectively,werereplacedbyAlaresidues,andinmutantEGFP:P6m3,theresiduesoftheEKImotifatpositions11to13wassubstitutedbyanAlatriplet.IncontrastwithEGFP:P6m1,whenexpressedinBY-2cellsbothconstructionswereexclu-sivelyfoundinthecytoplasm;no uorescencecouldbedetectedinthenucleusevenafter48to72hincubationofthetransfectedtobaccocells(Figure5B,panels7to10).However,EGFP:P6m2alsogeneratednumeroussmall uorescentfocithatweresuper-imposedonthediffuse uorescence(Figure5B,panel7;seealsoFigure8C,panel5),whereasEGFP:P6m3wasunabletoformaggregates.

Takentogether,theseresultsdemonstratethattheN-terminala-helixofP6isessentialfortheformationofviroplasmsand,furthermore,thatitsLeuzippermediatestheP6–P6interaction.Variousaminoacidsofthea-helix,notallofwhicharelocatedattheinterfaceofthepredictedcoiled-coilstructure(Figure4B),areimportantfortheaggregationprocess.Ifwerefertoourmodel(Figure4B),GlufromtheEKImotifandLeuatpositions14and18aredirectlyinvolvedintheinteractionbetweenP6molecules,thusexplainingtheinabilityofEGFP:P6m1andEGFP:P6m3toformaggregates.Whethertheotherresidues,inparticularthoselocalizedbetweentheLeuresidues,affectthe

secondary

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

CaMVP6IsaNucleocytoplasmicProtein933

Figure4.Fine-ScaleMappingoftheP6–P6InteractionDomain.

(A)SchematicrepresentationofdomainA(aminoacids1to112).TheconservedregioncanbedividedintotwosubdomainsdesignatedA1(aminoacids1to83)andA2(aminoacids84to112).Thetwoinvariantsequences(I1andI2)indomainAareindicatedbysolidlinesandthea-helixbyagraybox.aa,aminoacids.

(B)Top:thesequenceofaminoacids4to31ofP6containsfourtypicalLeuzipperheptadmotifs.Hydrophobicresiduesatheptadpositionsaanddareinbold.TheinvariantsequenceI1andthenearduplicatesequencei1areindicated.LeuresiduessubstitutedinEGFP:P6mbyaGln(atpositions14and16)andbyaHis(atposition18)arespeci ed.Bottom:acomputer-generatedmodelofaparallelcoiled-coilstructureformedbetweentheNterminioftwoP6molecules.Thesidechainsoftheresiduesinpositionsaanddareshown.

(C)GST:A,GST:A1,andGST:A2aswellasGSTandfull-lengthP6wereexpressedinE.coliandsubmittedtofarproteingelblotanalysisusing32P-labeledP6,GST:A,GST:A1,orGST:A2asprobeintheoverlay.Theradioactivecomplexesweredetectedbyautoradiographyafteranexposureof24h.Molecularmassesofmarkerproteinsareindicatedattheleft.

structureoftheNterminusofP6orthecompleteproteinremainsanopenquestion.Indeed,EGFP:P6m2couldstillgivesmallaggregatesbutlostthecapacitytoassembleintolargeperinu-clearviroplasms.

P6IsaNucleocytoplasmicShuttlingProtein

Asmentionedatthebeginning,smallaggregatesofEGFP:P6werefoundinthenucleusofBY-2cells(Figure2,panels4and5)andEGFP:P6DAwaspresentmainlyinthenucleus(Figure3B,panels1and2),althoughthecorrespondingfusionproteinshaveapproximatemolecularmassesof85and75kD,respectively,whicharehigherthanthereportedlimitforpassivediffusion

¨rlichandKutay,1999).We rstsus-acrossnuclearpores(Go

pectedthatEGFP:P6andEGFP:P6DAmightbecleavedbyacellularproteasetoproduceaspeciessmallenoughtofreelydiffusethroughthenuclearpores.SuchahypothesiswasconsistentwiththeobservationthataP6-speci cdegradationproductof42kDisfrequentlyfoundinCaMV-infectedplants(Mauleetal.,1989)andinheterologousexpressionsystems(Lehetal.,2000).However,proteingelblottingassaysperformedwithproteinsfromtransfectedBY-2cellsexpressingEGFP:P6orEGFP:P6DA,usingantibodiesraisedagainstGFP,revealedonlypolypeptidesof85and70kD,respectively(seesupplemen-taldataonline),indicatingthatnosigni cantdegradationofthefusionproteinsoccurred.Consequently,EGFP:P6andEGFP:P6DAareprobablytransportedactivelyintothenucleusoftobaccocells.

Theaforesaidobservationsraisedthequestionwhetherfull-lengthP6mightlikewiseenterthenucleusofhostplantsduringCaMVinfection.Toanswerthisquestion,wepreparedproto-plastsfromsystemicallyCaMV-infectedandhealthyturnipplantsandperformedimmunodetectionofP6usinganti-P6andsecondaryantibodiescoupledtoAlexa488.Observationofthecytosolandinparticularthenucleusby uorescentmicros-copyunderstandardconditionswasoftenhinderedbythepresenceofnumerouschloroplasts.Nevertheless,diffuse uo-rescencecouldbevisualizedwithinthenucleusbutnotthenucleolus.Generally,theprotoplastscontainednumerousP6aggregatesinproximitytothenuclearmembranesothatitwasdif culttodeterminewhethersigni cantamountsoftheimmu-nolabeledP6wereindeedwithintheinteriorofthe

nucleus.

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

Figure5.TheN-Terminala-HelixofP6IsanEssentialDeterminantfortheFormationofViroplasms.

(A)SchematicrepresentationofEGFP:P6anditsdeletedversions.P6fragments:thenumberscorrespondtotheaminoacidpositionswithintheclonedP6sequence.EGFPanddomainAarerepresentedbygreenandyellowboxes,respectively(nottoscale).TheLeu-enrichedsequences(i1-I1andI1)areindicatedaboveEGFP:P6Di1-I1andbelowEGFP:P6DI1,respectively.I1indomainAisrepresentedbyabluebox.Theemptyspaceandtheredstarintheblueboxindicatethedeletionandthepointmutations,respectively.TheotherdomainsofP6arerepresentedasinFigure1.AminoacidsmutatedinmotifI1ofthethreeEGFP:P6mversionsaredepictedinred.Theothermotifsarede nedinFigure1.LegendCLSMimages:numberingrefersto(B).aa,aminoacids.

(B)SubcellularlocalizationofpointmutatedEGFP:P6anddeletedversions(images1to10).The uorescentproteinswereexpressedinBY-2cellsandthe uorescentimages(1,3,5,7,and9)collectedbyCLSMweresuperposedonthecorrespondingdifferentialinterferencecontrastimages(2,4,6,8,and10).Bars¼10mm.

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

Figure6.PointMutationsintheInvariantSequenceI1ImpairP6–P6Interactions

Proteinextractsofbacteriaexpressingeitherwild-typeP6(laneP6),point-mutatedP6(laneP6m1),oranextractofbacteriatransformedwithemptyvector(laneE.coli)werefractionatedbySDS-PAGE(15%)andtransferredontoanitrocellulosemembrane.ThemembraneswereincubatedeitherwithantibodiesraisedagainstP6(rightpanel)orsubmittedtoafarproteingelblotassayusinginvitro32P-labeledP6(leftpanel).Molecularmassesofmarkerproteinsareindicatedtotheleft.

Therefore,immunolabelingwithanti-P6antibodieswasper-formedonpuri ednucleiisolatedfromhealthyandCaMV-infectedturnipprotoplasts;thenucleiwerealsostainedwithpropidiumiodide(Figures7Aand7B,panel2).Nucleipreparedfromhealthyplantsneverreactedwithanti-P6antibodies(Figure7A),whereas;50%ofthosefrominfectedplantswereimmu-nolabeled(Figure7B).P6-Alexa488 uorescentfociwereobservedinthenucleoplasmand/orthenucleolusfrominfectedturnipcells,thusdemonstratingthatP6moleculesdoindeedenterthenucleusduringtheCaMVinfectioncycle.ThenucleiwereoftencontaminatedwithP6-containingviroplasmsbe-causethelatterremained rmlyattachedtotheoutersurfaceevenwhentheywerefurtherpuri edthroughacompositesucrose/Percollgradient.Therefore,werealizedaseriesofphotographsobtainedbyCLSManalysisof0.5-mm-thicksec-tionsacrosspuri edandP6-immunolabelednuclei.AsshowninFigure7C,largecontaminatingviroplasmsprogressivelydisap-pearedfromviewinsuccessivesections(panels5to14),whereassmallP6-labeledaggregatesprogressivelyappearedwithintheorganelle(panels13to18),thusprovidingadditionalevidenceforthepresenceofP6withinthenucleus.The uores-centfocimightcorrespondtoP6aggregatesand/ortointer-actionsbetweenP6andspecializednuclearcompartmentssuchasspeckles(forareview,seeLamondandSpector,2003)orCajalbodies(Ochsetal.,1994).ThelocalizationofP6inthenucleolus,observedinsomecases,mightberelatedtoitscapacitytointeractwiththeribosomalsubunitsasshownbyParketal.(2001).

ToexcludethepossibilitythatP6enteredthenucleiduringtheirpuri cation(i.e.,bydiffusionthroughanalterednuclearenvelope),nucleifromhealthyprotoplastswereincubatedduring30minat48Cwith;100mgofsolubleP6proteinexpressedinE.coli.Thepreparationwasimmunolabeledasdescribeabove.No uorescencewasdetectablewithintheorganellesafterthistreatment,thusreinforcingourconclusionthattheP6proteinfoundinnucleifrominfectedcellsisactivelytransportedthereinthecourseoftheCaMVreplicationcycle.

CaMVP6IsaNucleocytoplasmicProtein935

Inconclusion,ourdataclearlysupporttheideathatP6canenterthenucleusduringviralinfectionandalsoindicatethatP6isanucleocytoplasmicshuttlingprotein.Furthermore,our ndingsalsostronglysuggestthatthesequencedownstreamofdomainAisimplicatedinthenuclearlocalizationofP6,whereastheN-terminalregionofP6mightcontainanuclearexportsignal(NES).

TheNTerminusofP6ContainsanNESThatIsRecognizedbytheCRM-1NuclearExportPathway

ThelatterhypothesisisreinforcedbythefactthatdeletionoftheconservedhydrophobicsequenceI1locatedinsubdomainA1ormutationoftheLeuresiduesatpositions14,16,and18partiallyabolishednuclearexportofP6(Figure5B,panels3to6).Moreover,thisLeu-richsequencebearssomeresemblancetotheNES(EKDTLLIDL)foundintheBR1proteinoftheSquashleafcurlvirus,ageminivirus(WardandLazarowitz,1999),andtotheNESsequenceofseveralknownrapidlyshuttlingnuclearpro-teins,suchasHIVRevprotein(forareview,seePollardandMalim,1998).

ToprovidefurtherevidencethattheaforesaidsequenceisanNES,wedeletedthesequenceI1ormutatedLeuresidues14,16,and18intheEGFP:Afusionprotein(Figure8A)asdescribedabove,andthebehaviorintobaccoBY-2cellsofthemutantswascomparedwiththatofthenonmutatedprotein.TheseexperimentswereperformedwithEGFP:Aratherthanwiththefull-lengthP6becausenuclearaccumulationofEGFP:Amutantswouldbedirectlyrelevanttotheimpairmentoftheexportprocess,whereastheaccumulationofEGFP:P6mutantsinthenucleusdoesnotpermitdiscriminationbetweenanexportdefectandanactiveimportoftheprotein.Indeed,thewild-typefusionproteinEGFP:Awasneverfoundinthenucleus,althoughitisofasizethatshouldpermitittodiffusefreelythroughthenuclearpore(Figure3B,panels3and4).

WhensequenceI1wasremovedfromdomainA,EGFP:ADI1wasequallydistributedinthecytoplasmandthenucleusexceptforthenucleolus(Figure8B,panels1and2),whereasEGFP:Alocalizedexclusivelytothecytoplasmiccompartment(Figure3B,panels3and4).ThesubcellularlocalizationinBY-2cellsofmutantEGFP:Am(Figure8B,panels3and4),inwhichthethreeLeuresiduesofI1werereplacedbypolarresidues,wassimilartothatobservedwithEGFP:ADI1(Figure8B,panels1and2);EGFP:Amwasfoundinboththecytoplasmandinthenucleusbutnotinthenucleolus.TheabsenceofbothEGFP:Amutantsinthenucleolus,incontrastwiththesituationwithEGFP:P6DI1andEGFP:P6pointmutatedversions(Figure5),mightbeduetothefactthattheN-terminalregionofP6isunabletointeractwithribosomalproteins,whereastheEGFP:P6mutantsstillcontainthecorrespondinginteractiondomains(i.e.,mini-TAVandRNAbindingdomainA)(Lehetal.,2000;Parketal.,2001;Bureauetal.,2004).

Alltheseresultssupportamodelinwhich(1)bothEGFP:AandEGFP:Amutantscanenterandexitthenucleusbydiffusion,(2)EGFP:AmoleculesbutneitherEGFP:ADI1norEGFP:Amarerapidlyexportedtothecytoplasm,and(3)thatthesequenceI1functionsasanNESbecausepointmutationsofLeuresiduesinI1impairtheexportofEGFP:A.Thus,theseLeuresidues

appear

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

936ThePlantCell

Figure7.NuclearLocalizationofP6.

Nucleifromhealthy(A)andCaMV-infectedturnipleaves(B)were xedandimmunolabeledwithrabbitanti-P6antibodiesandmouseanti-rabbitIgGcoupledtoAlexa488assecondaryantibodies(image1)andstainedwithpropidiumiodide(image2).Panel3correspondstodifferentialinterferencecontrastimagesandtheright-handimages(panel4)totheirsuperpositionwiththe uorescentandpropidium-stainedimages.Theconfocalimageswerecollectedwithafocaldepthof0.45mm(C).Aseriesofsuchopticalsectionsthroughanucleusisolatedfromaninfectedplant,anti-P6immunolabeled,andstainedwithpropidiumiodide.Bar¼5mm.

tobeessentialresiduesforthenuclearexportofP6asalreadydescribedfornucleocytoplasmicshuttlingproteinspossessingaLeu-richNESandinparticularfortheBR1proteinofSquashleafcurlvirus(WardandLazarowitz,1999).However,wecannottotallyexcludethattheinvariantsequenceI1alsohaspropertiesinvolvedintheretentionoffusionproteininthecytosol.

Therefore,thenuclearexportofP6wasfurtherinvestigated,treatingBY-2cellstransfectedwiththeaforementionedre-combinantplasmidswithleptomycinB,whichspeci callyinhib-itstheCRM-1pathwayinvolvedinthenuclearexportofmanyproteins(Fornerodetal.,1997;Kudoetal.,1998).WhenBY-2cellsexpressingEGFP:Awereincubatedwith100nMlepto-mycinB,6hafterbombardment,the uorescentproteinaccu-mulatedabundantlyinthenucleus(Figure8C,panel2),whereasno uorescencewaspresentinthiscompartmentinuntreatedcontrolcells(Figure8C,panel1),thuscon rmingthatEGFP:Amoleculesareactivelyexportedfromthenucleus.ThefusionproteinEGFP:P6formedlargeaggregatesinthecytoplasmandwasundetectableinthenucleusoftransfectedtobaccocells(Figure8C,panel3),butwhenthelatterweretreatedwithleptomycinB,itwasfoundinboththecytoplasmandinthenucleus(Figure8C,panel4).Thenuclearcompartmentcon-taineddiffuse uorescence,accompaniedasexpectedbymanysmallaggregatesbecauseP6wasabletoself-interact.ThisresultprovesthatP6isactivelytransportedbetweenthenucle-ocytoplasmiccompartments.SimilarexperimentswerealsoperformedwithpointmutatedEGFP:P6versionstodeterminethefunctionalimportanceofdifferentresiduesinthenuclearexportofP6.Asexpected,themutantEGFP:P6m1hadthesamesubcellularlocalizationinBY-2cellstreatedwithleptomycin

B

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

Figure8.TheLeu-EnrichedSequenceofDomainAIsResponsiblefortheNuclearExportofP6.

(A)SchematicrepresentationofEGFP:Aandmutatedversions.EGFP,domainA,andmotifI1ofP6arerepresentedasinFigure5.TheemptyspaceandtheredstarindicatethedeletionandthepointmutatedmotifI1,respectively.

(B)SubcellularlocalizationinBY-2cellsofEGFP:AI1andEGFP:Am(images1to4).The uorescentimages(1and3)observedbyCLSMaresuperposedonthecorrespondingdifferentialinterferencecontrastimages(2and4).

(C)EffectofleptomycinBonthesubcellularlocalizationofEGFP:Afusionproteinsandofwild-typeandpointmutatedformsofEGFP:P6.FluorescentcellswereselectedunderanHBOlamp,collectedinBY-2cellculturemediumcontaining100nMleptomycinB(imagestotheright)ornoleptomycin(imagestotheleft),andincubated6hat248Cwithgentleshaking.Thepoint-mutatedaminoacidswithintheI1sequenceareindicated.Bars¼10mm.

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

938ThePlantCell

(datanotshown)ornot(Figure5B,panels5and6)becausemutationsoftheLeuresiduesaresuf cientpersetoimpairtheexportofthefusionprotein.The uorescenceinthecytoplasmevenafterleptomycinBtreatmentsuggeststhatnotallEGFP:P6m1moleculesenteredthenucleusbecausetheywereretainedinthecytoplasmor,alternatively,thatotherresiduesoftheI1sequencemightcontributetotheexport.IncontrastwithEGFP:P6m1,leptomycinBhadonlylittleeffectonthenuclearexportofEGFP:P6m2becausealmostno uorescencecouldbedetectedwithinthenucleus(Figure8C,panels5and6)inmostBY-2cellsobservedbyCLSM;onlyfewcellsexhibitedahighly uorescentnucleus.ThedifferenceobservedintheresponsetoleptomycinBtreatmentmightbeduetothefactthat(1)theresiduesmutatedinEGFP:P6m2areindeedessentialforexportashypothesizedpreviouslyor(2)thattheirmutationsmodifytheconformationofEGFP:P6inamannerthatinterferessomehowwiththeactivityoftheinhibitor.TheEGFP:P6m3mutantmainlyaccumulatedinthenucleoplasmwhenthecellswereincubatedinthepresenceofleptomycinB,whereasitdidnotinuntreatedcells;onlylowlevelsofEGFP:P6m3werefoundinthecytoplasmoftreatedcells(Figure8C,panels7and8).ThisresultsuggeststhattheEKImotifisnotpartoftheNES,orifitis,ithasaminorin uenceontheexportofP6bytheCRM-1pathway.

Together,thesedataleadtotheconclusionthatP6containsatitsNterminusanNESandthatitsLeuresiduesinvolvedinthenuclearexportofP6arerecognizedbytheCRM-1pathway.TheactivityofthisexportpathwaylimitstheaccumulationofP6inthenucleusthatcouldbedeleteriousfortheCaMVinfectiouscycle.DISCUSSION

DuringthecourseofaCaMVinfection,P6formselectron-denseviroplasmsthatarethoughttobevirionfactoriesthatserveasascaffoldforvirusreplicationandassembly.Inthisstudy,wehaveinvestigatedbyinvitroandinvivoexperimentalapproachesthemechanismleadingtotheformationoftheseviroplasms.OurresultsshowthatP6self-interacts,asalreadysuggested(Haasetal.,2000),andfurtherdemonstratethat,atleastinvitro,theP6–P6interactionsareexclusivelymediatedbytheN-terminalregionencompassingresidues1to83(subdomainA1).NootherP6sequencewasabletointeractinvitrowithfull-lengthP6.ThefactthatfusionofdomainAtounrelatedproteinsdrovethisinteractioninvitrodemonstratesthattheNterminusofP6issuf cientpersetopromoteself-interactionindependentlyfromneighboringsequences.OurresultsonlypartiallyagreewiththoseobtainedbyLiandLeisner(2002),whousedtheyeastdoublehybridsystemtoshowthat,inadditiontotheNterminus,threeotherdomainsofP6areabletobindfull-lengthP6,namelythemini-TAVdomain,thedownstreamadjacentsequence,andtheC-terminalregionexceptfora90-amino-acid-longse-quence.Possiblytheadditionalinteractingdomainscharacter-izedinthedoublehybridsystembindtoP6withanaf nitythatistoolowfordetectioninfarproteingelblotexperiments.In-completerenaturationofthedomainsinquestioncouldalsointerferewithactivityintheinvitroassays.Ontheotherhand,wecannottotallyexcludethepossibilitiesthatsomeoftheinter-actionsdetectedinyeastbyLiandLeisner(2002)mightbemediatedbyyeastRNAorproteins.Indeed,domainsofP6identi edbytheseauthorstobeinvolvedinP6–P6interactionshavenucleicacidbindingpropertiesand/orinteractwithcellularproteins,inparticularnuclear-localizedproteins(Parketal.,2001;Bureauetal.,2004).

TheroleofsubdomainA1intheformationofviroplasmswasinvestigatedbytransientexpressionintobaccoBY-2cellsoffull-lengthP6anddeletedversionsofP6fusedtoEGFP.Expressionoffull-lengthP6fusionproteingaverisetoviroplasmslocatedintheproximityofthenucleus,whicharestructurallysimilartothosefoundinCaMV-infectedplants.Indeed,inbothcases,theviroplasmsappearedbyconfocalmicroscopyanalysisasag-gregatesofmultiplehollowmacromolecularstructures,anditissuggestedthattheymightbeproducedstepwisebytheassem-blyofthedonut-likestructuresthatcouldbevisualizedinsometransfectedtobaccocells.SimilarhollowstructureswerealsoobservedwhenNSP2andNSP5proteins,thetwomajorviralcomponentsofdenseviroplasmsinducedbyrotaviruses,werecoexpressedinculturedhostcellsintheabsenceofotherrotaviralproteinsandofrotavirusreplication(Fabbrettietal.,1999).WeassumethatthehollowcenteroftheP6aggregatescorrespondstotheelectron-lucentholesofviroplasmsobservedbyelectronmicroscopyinCaMV-infectedcells(Xiongetal.,1982).

TheresultsobtainedbytransientexpressionoftruncatedversionsofP6clearlydemonstratethat,althoughthe83N-terminalaminoacidsofP6arenecessary,theyarenotsuf -cientfortheformationofviroplasmsandthatconsequentlyotherregion(s)ofP6areimplicatedinthisprocess.Thedomainbetweenaminoacids289and379,referredtoasD3byLiandLeisner(2002),mightbeoneofthesesequencesbecausetheseauthorshaveshownthatitplaysanimportantroleinP6self-associationwhentestedinvivousingtheyeasttwohybridsystem.ThisregionofP6isalsoengagedininteractionsneededfortranslationaltransactivation(DeTapiaetal.,1993;Parketal.,2001).Inanyevent,our ndingssuggestthattheN-terminallymediatedP6–P6interactionisaprerequisiteforfurtherinter-actionsbetweenotherP6sequencesand/orforstabilizationofmacromolecularstructuresbecauseformationofviroplasmswastotallyimpairedwhenanN-terminallytruncatedP6(P6DA)wasexpressedintobaccocells.

ComputeranalysisofsubdomainA1indicatedthatitformsanamphipathica-helixatitsNterminus(residues4to31).ThelattercontainsaLeuzippermotifthatcouldformaparallelcoiled-coilstructure(Lupas,1997),stronglysuggestingthatsuchaconfor-mationisimplicatedintheinteractionbetweenP6molecules.Thishypothesisiscon rmedbytheresultsoffarproteingelblotassaysshowingthattheintermolecularinteractionislostifkeyhydrophobicaminoacidsoftheLeuzippermotifsaresubstitutedbypolarresidues.InvolvementofsuchaninteractionintheformationofviroplasmsisevidencedbythefailureofP6carryingthesepointmutationsinthea-helixtoformaggregatesintransfectedtobaccocells.Currently,wehypothesizethatthecoiled-coilformationbetweentheNterminiofinteractingP6moleculesinducesconformationalchangesthatallowotherregionsofP6toparticipateintheaggregationprocess.Indeed,mutationsofaminoacidsthatarenotlocatedatthecoiled-coilinteractingsurfacedidnotpreventtheformationofsmall

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

CaMVP6IsaNucleocytoplasmicProtein939

aggregates;however,theyimpairedassemblyoftheseaggre-gatesintoviroplasms.Itisevidentthatafurtherunderstandingofthemechanismofviroplasmformationwillalsorequirecharac-terizationoftheotherdomainsofP6implicatedinthisprocessaswellaspossiblecellularstructuresand/orfactorsthatmightbeinvolved,suchasendomembranesand/orthemicrotubularnetwork.Nevertheless,nohost-speci cfactorsseemtobeneededbecauseviroplasmsthatformedintobaccocells,anon-hostforCaMV,aresimilartothosefoundinhostcells.Asnotedearlier,membranecomponentsmayfunctionintheearlystepsofP6self-assembly,characterizedbyformationofsmallaggregatesasthelatterdisappearedupontreatmentoftobaccocellswithnonionicdetergent,whereastheviroplasmslocatedatthenuclearperipherywereunaffected(datanotshown).Furtherinvestigationswillberequiredtodeterminewhetherbindingtoendomembranesisessentialfortheself-assemblyofP6andwhetheritshydrophobicNterminus,whichpresentsfeaturesofapeptidesignal(BlobelandDobberstein,1975),playsarole.Becauseviroplasmformationdoesnotvisuallyperturbthemicrotubuleortheactin lamentnetworksinBY-2cells,itwillbeofparticularinteresttodeterminewhetherviroplasmformationinvolvesthecytoskeleton,asdescribedinanimalcellsforaggresomesandfortheviralfactoriesinducedbylargecytoplasmicDNAvirusessuchasAfricanswinefevervirus(Kopito,2000;Heathetal.,2001).Johnstonetal.(1998)pro-posedamodelinwhichsmallaggregatesaredeliveredbyretrogradetransportalongmicrotubulestotheperipheryofthenucleuswheretheyareassembledintolargestructures.

TransientexpressionofEGFP-taggedP6ledtotheunex-pecteddiscoverythatP6,considereduntilnowasacytoplasmicprotein,isactuallyanucleocytoplasmicshuttlingprotein.Thepresenceofthisviralproteinwithinthenucleuswascon rmedbyitsimmunodetectioninnucleipreparedfromCaMV-infectedturnipleavesandCLSM-generatedopticalserialsectionsthroughtheorganelles.Ourresults,obtainedwithtransfectedtobaccocells,indicatethatonlyasmallfractionofP6,probablymoleculesthatarenotengagedintheaggregationprocess,entersthenucleus.Indeed,EGFP:P6mainlyformedinclusionbodiesinthecytoplasmandwasalmostundetectableinthenucleusoftobaccocells.OnlyinhibitionoftheexportprocessbyleptomycinBsystematicallypermittedobservationofdiffuseEGFP:P6andsmallaggregateswithinthenucleus.OurresultsalsoindicatethattheexportofP6probablyoccursveryrapidlyininfectedcells,sothatonlylowamountsarepresentinthenucleusatanytime.ThestrongP6nuclearexportactivityprobablypreventsaccumulationofP6withinthenucleus,whichcouldbedeleteriousforCaMVinfectivity.Inaddition,itismorethanlikelythatthenucleocytoplasmictransportis nely

Table1.OligonucleotidesUsedasPrimerstoGeneratethePCRProductsClonedintopGEX-2TK,pCK-EGFP,pETP42,andpETKaKS.6VectorsNameA

ABam(þ)AEco(ÿ)A1Eco(ÿ)A2Bam(þ)A2Eco(ÿ)B

Bsrmut(þ)Bsrmut(ÿ)P6Bsr(þ)PBsr(þ)P6DABsr(þ)QBsr(þ)P6Xba(ÿ)AXba(ÿ)A1Xba(ÿ)BXba(ÿ)QXba(ÿ)C

ANco(þ)ANco(ÿ)D

NESmut(þ)DNES(ÿ)NES(þ)Di1(ÿ)

Sequence

CloningofsequencescorrespondingtoCaMVP6truncatedversionsgccggatccATGGAGAACGAAAAACTCgatgaattcTCATGGAATTCCCTGATGAGGcacgaatccCTAAGCCATCAACGGATTTGggcggatccTCCAATATCTTGTCAAAAGATcacgaattcCTATTCTGCTCTGAGAGGAGC

CloningofsequencescorrespondingtoCaMVP6proteinanditstruncatedversions

GACTGGGGTTGTACTAAGGCCGCCTTAGTACAACC

catgtacaagATGGAGAACATAGAAAAACTCATAGTACAACCgcatgtacaagCTCAAGATCAGAAGTACTATTCgcatgtacaagATCCCACAAAAATCTGAGCTTAAgcatgtacaagCCAATCCCACAAAAATCTGAGgattctagaTCAATCCACTTGCTTTGAAGACgattctagaTCATGGAATTCCCTGATGAGGgattctagaTCAAGCCATCAACGGATTTGTgattctagaTCAGGAGATCTCTTTTGGGGC

gattctagaTCAAAATATGTCTTTCTCTGTGTTCTTG

CloningofthesequencecorrespondingtotheP6N-terminaldomain(aminoacids1to112)

gatccatggATGGAGAACATAGAAAAACTCctaccatggtAATTCCCTGATGAGGACGSite-directedmutagenesis

AAAATACAAATGCAAGAACACGATCTACTCTTGCATGAGGAGTTTTTGTAAGAGCAAAAATAAGCTTATATGTTCTCCATCTTGTACAGC

RestrictionSiteBamHIEcoRIEcoRIBamHIEcoRI

BsrGIBsrGIBsrGIBsrGIXbaIXbaIXbaIXbaIXbaI

NcoINcoI

Forwardprimer(þ)andreverseprimer(ÿ).Restrictionsitesintheprimersequencesarerepresentedbybold-facedlower-caseletters.PCRproductswereclonedintopGEX-2TK(A),pCK-EGFP(BandD),pETP42(C),andpETKaKS.6(D)vectors.

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

940ThePlantCell

regulatedduringtheviralcycle(inotherwords,thatitoccursonlyatspeci cstages).Thesewouldexplainthedif cultyween-counteredtodetectP6innucleifrominfectedplants.

ThestudyofthebehaviorofP6mutantsinBY-2cellsrevealedthatthenuclearexportactivityisassociatedwiththeLeu-richsequence(residues11to20)attheNterminusofP6.Itsin-volvementinnuclearexportwasdemonstratedbytheincapacityofP6toexitthenucleuswhenLeuresiduesofthesequencewerepointmutatedandbythefactthatmutateddomainAofP6accumulatesinthenucleus,incontrastwiththewild-typeform.ThesequenceEKIisnotimplicatedintheexportofP6asevidencedbytheresultsofexperimentsperformedwithlepto-mycinB,butitisanimportantdeterminantfortheformationofviroplasms.ConcerningtheinvolvementoftheotherresiduesoftheI1invariantsequence,furtherinvestigationsarenecessarytode nitivelyanswerthisquestion.Interestingly,theNESispartofthea-helixthatisinvolvedinP6self-assemblyandthisfactmightexplainwhydeletionofthe rst90nucleotidesoftheCaMVORFVIabolishessystemicinfectionandsigni cantlyreducesthereplicationofthegenomeinsinglecells(KobayashiandHohn,2003).TheoverlapbetweendomainsinvolvedinP6exportandself-assemblyalsoraisesthequestionofhowthesetwoactivitiesareregulatedduringtheviralcycle.WehypothesizethatP6proteinshuttlingbetweenthenuclearandcytoplasmiccompart-mentsprimarilyinvolvesapopulationofP6monomers(ordimers)thathaveescapedtheaggregationprocess.Recentstudieshavedemonstratedthatimportinsful lladualfunctionasanuclearimportreceptorandcytoplasmicchaperonefornuclearimported

proteins(Ja

¨keletal.,2002).SuchanantiaggregationmechanismmightalsobeinvolvedforP6molecules.Thisdoesnot,however,excludethepossibilitythatP6couldbeincorporatedintoviroplasmsaftertheirexportfromthenucleus.

ThediscoverythatP6isanucleocytoplasmicshuttleproteinopensnewprospectsforunderstandingthemechanismsbywhichthisviralproteinregulatestheCaMVinfectiouscycle.Thefunction(s)ofP6inthenucleuscanonlybeamatterforspeculationatpresent.P6mighthavearolesimilartotheRevproteinofHIV-1(PollardandMalim,1998)incontrollingexportofCaMV35SRNAanditssplicedversionsbecauseitalsohasthecapacitytobindsingle-anddouble-strandedRNA(DeTapiaetal.,1993;Cerritellietal.,1998).ThepresenceofP6inthenucleolus,whereassemblyofribosomalsubunitsoccurs,raisesthepossibilitythatP6mightinteractdirectlywithribosomesbeforetheirexporttorenderthemcompetentfortranslationoftheCaMVpolycistronicmRNA.TheribosomalproteinsL18andL24,whichinteractwiththemini-TAV(Lehetal.,2000)andRNAbindingdomains(Parketal.,2001)ofP6,respectively,couldbetargetsforP6becausetheyparticipateintheformationofthe60Ssubunitinthenucleolus(Andersenetal.,2002).OtherfunctionsmightalsobeassociatedwiththenucleocytoplasmiclocalizationofP6(i.e.,inhibitionofnonsense-mediatedmRNAdecaytopreventdegradationofthe35SRNAanditssplicedversions)(forareview,seeMaquatandCarmichael,2001).Thesehypothesesaresupportedbythe ndingthatP6nuclearexportismediatedbytheCRM-1pathway(Kudoetal.,1998),whichisknowntobespeci callyusedforexportoftheribosomalsubunitsandofsomecellularmRNAs(forareview,seeWeis,2002).

METHODS

ConstructionofRecombinantPlasmids

RecombinantplasmidswereconstructedbyinsertionofviralsequencesintothepET3aderivativespETKaKS(Lehetal.,2000),pGEX-2TK(Amersham-PharmaciaBiotech,Uppsala,Sweden),andpCK-EGFP(Clontech,PaloAlto,CA).DNAfragments ankedbyappropriatere-strictioncloningsitesweregeneratedusingPCR;theoligonucleotidesusedforPCRarelistedinTable1.

CaMVORFVIanditsderivativeswereclonedeitherintotheKpnIandSacIsitesorintotheSacIsiteofthepETKaKSplasmid.ViralDNAsequenceswereampli edfromplasmidpMD324containingtheCaMVCabb-JIgenome(DelsenyandHull,1983)usingtwoprimersbearingattheir59terminiKpnIandSacIsites,respectively,orSacIsites.TheDNAfragmentsweredigestedwiththeappropriaterestrictionenzymesandintroducedintopETKaKScleavedwithKpnIandSacIorwithSacI.Allconstructswerecon rmedtobeerrorfreebysequencing.ExpressionoftherecombinantplasmidsinEscherichiacoligeneratesfusionproteinscontainingattheirNterminusthedecapeptideMet-Arg-Arg-Ala-Ser-Val-Gly-Ser-Gly-Thr,whichcanbephosphorylatedinvitrobyaproteinkinasefrombovineheartmuscle(thephosphorylationsiteisinbold-facedtype).

TheDNAsequenceencodingtheNterminusoftheCaMVP6protein(nucleotides1to336ofORFVI)wasampli edfromthepETKaKS.6recombinantplasmidencompassingthecompleteORFVIusingprimerscarryinganNcoIrestrictionsiteattheir59end.ThePCRfragmentwasdigestedwithNcoIandclonedintopETP42(Lauberetal.,1998),whichhadbeencleavedwiththesameenzymetoproducepET-A:P42.PlasmidspGST:A(ORFVInucleotides1to336),pGST:A1(ORFVInucleotides1to249),andpGST:A2(ORFVInucleotides250to336),codingfordifferentregionsoftheP6NterminusfusedtoGST,wereobtainedbyPCRampli cationofdifferentORFVIsequenceswithprimerscontaining59terminalBamHIandEcoRIsites(Table1).Theampli edDNAfragmentsweredigestedwiththeappropriaterestrictionendonucleasesandclonedintolinearizedpGEX-2TK.

ThepCK-EGFPvectorwasusedtoconstructtherecombinantplas-midscodingforfusionproteinsbetweenEGFPandwild-typeCaMVP6orP6mutants.ThecorrespondingORFVIsequenceswereampli edbyPCRfrompMD324usingtwoprimerscarryingattheir59endsBsrGIandXbaIsites,respectively(Table1).

DeletionsandpointmutationswereintroducedinEGFP:P6andEGFP:Abysite-directedmutagenesis(Stratagene,LaJolla,CA).Therecombinantplasmidswereampli edbyPCRusingpfuTurbopoly-meraseaccordingtothemanufacturer’sinstructionsanddesignedinternaloligonucleotidesasprimers(Table1).ThemixturewasthenincubatedwithtwounitsofDpnIfor2hat378Ctodestroythetemplate.The59endsofPCRproductswerephosphorylatedbyT4polynucleotidekinaseinthepresenceof1mMATPforsubsequentligation.Error-freerecombinantplasmidswereidenti edbyDNAsequencing.

ProductionandPhosphorylationofRecombinantProteins

E.coliBL21/DE3(pLysS)strainwastransformedbyelectroporationwithpETKaKSandpGEX-2TKrecombinantplasmidscodingforfull-lengthP6orP6mutants.Expressionoftheheterologousproteinswasinducedwith1mMisopropylb-thiogalactosidefor2honcethebacterialculturehadreachedtheexponentialphase.Bacteriawerecollectedbycentrifu-gationat5000gfor5min,resuspendedinHMKbuffer(20mMTris-HCl,pH7.5,100mMNaCl,and12mMMgCl2),andlysedbysonication(threepulsesfor20sat50W).Aftercentrifugationat12,000gfor10min,thesupernatantwasdiscardedandtheinclusionbodiesresuspendedin500mLofHMKbuffer.

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

Full-lengthP6andtheP6fragmentsA,A1,andA2expressedfrompETKaKSandpGEX-2TKvectors,respectively,werelabeledinthepresenceof[g-32P]ATP(3000Ci/mmole)andbovineheartmuscleproteinkinase(10units)for2hatroomtemperature,accordingtotheinstructionsofthemanufacturer(Sigma-Aldrich,St.Louis,MO).ExcessradioactiveATPwaseliminatedby ltrationthroughaSephadexG50orG25column(Amersham-PharmaciaBiotech)dependingonthemolecularmassofthelabeledfusionproteins.ProteinGelBlotAnalysis

ProteinsfromrecombinantbacteriawereseparatedbySDS-PAGEandelectrophoreticallytransferredontoanitrocellulosemembrane(SchleicherandSchuell,Dassel,Germany).Themembraneswereblockedovernightin5%nonfatdriedmilkinPBSbuffer(140mMNaCl,2.7mMKCl,and8.1mMNa2HPO4,pH7.3)containing0.1%Tween20andthenincubatedfor4hatroomtemperaturewithspeci crabbitorsheeppolyclonalantibodiesraisedagainstP6(1:10,000dilution)orGST(1:5,000dilution),respectively.ThemembraneswerewashedwithPBSbufferandtreatedwithgoatanti-rabbitIgGantibodies,respectively,conjugatedeithertoalkalinephosphataseorperoxidase,atthedilutionrecommendedbythemanufacturer.FarProteinGelBlotAssays

Aproteinblottingoverlaytechniquewasusedtodetectinteractionsbetweenproteins.ProteinswereresolvedbySDS-PAGEandtransferredontoanitrocellulosemembrane.Membraneswerewashedseveraltimesat48CinHMbuffer(10mMTris-HCl,pH7.5,100mMNaCl,and25mMMgCl2)containing5%nonfatdriedmilkandincubatedfor12hat48Cwithgentleshakinginthesamebuffercontainingthe[32P]-labeledproteinintheoverlay.AfterthreewashesinHMbuffer,themembranesweredriedandradioactivecomplexesweredetectedbyautoradiography.TransientExpressioninTobaccoBY-2Cells

TheCaMVP6proteinanditsdeletedversionsfusedtoEGFPweretransientlyexpressedinBY-2tobaccosuspensioncells(NicotianatabacumcvBrightYellow2)maintainedasdescribedbyBanjokoandTrelease(1995).Cellsweresubculturedeach7dandharvested3daftermediumrenewalforbiolistictransfection.Cellswere lteredontoWhatmandisksandplacedfor2to4hon0.8%agarMSmediaplatessupplementedwith0.1Mmannitoland0.1Msorbitol.Particleprepara-tionandbombardmentassayswereperformedasdescribedbyHunoldetal.(1995)withmodi cations:2mgof1.1mmtungstenparticles(Bio-Rad,Hercules,CA)wereimmersedin1mLofabsolutealcoholfor20min.Driedparticleswerethensuccessivelymixedwith10mgofrecombinantplasmidDNA(pCK-EGFPvector)supplementedwith18%glycerol,0.75MCaCl2,and90mMspermidineina nalvolumeof90mL.The r-ingdistancewas11cmandtheheliumpressurewas7bars.Afterbom-bardment,cellsweretransferredto0.8%agarMSmediaplatesandincubatedinthedarkat288C.BY-2transfectedcellswerecollectedunderHBObinoculars(excitation/emissionwavelength488/505to545nm)20hafterbombardmentandculturedinMSliquidmediumbeforefurthertreatmentand/orCLSMobservations.VirusandHostPlant

Turnips(BrassicarapacvJustRightF1hybrid,providedbyTakiiandCo.,Kyoto,Japan)weremechanicallyinoculatedatthefourleafstage(Jacquotetal.,1998)withCaMVCabb-JIandgrowninagreenhouseat228Cfor5weeksbeforepreparationofprotoplastsfrominfectedleaves.

CaMVP6IsaNucleocytoplasmicProtein941

IsolationofTurnipNuclei

ProtoplastspreparedfromCaMV-infectedandnoninfectedturnipleaves(Kobayashietal.,1998)wereusedtoisolatenuclei.Approximately83106protoplastswerewashedtwiceinnucleibuffer(250mMsucrose,25mMMes,0.5mMEDTA,1mMMgCl2,1mMEGTA,pH5.5,andacompletecocktailofproteaseinhibitors[Roche,Indianapolis,IN]).Aftercentrifugationfor5minat100g,theprotoplastswereresuspendedin50mLofcoldnucleibuffercontaining1mMDTT,0.025%NonidetP-40,and1mMphenylmethylsulfonyl uorideandshakenslowlyfor20minat48C.Nucleiwereisolatedby lteringthesuspensionthrough50-mmmeshnylonandcollectedat48Cbycentrifugationfor5minat550g.Theywereresuspendedinthenucleibufferandcentrifugedat48Cthroughadiscontinuousgradientcomposedof18%Ficolland85%Percollfor15minat8000g.Thebandcontainingthenuclei,locatedbetweentheFicollandPercolllayers,wasdilutedthreefoldwith10mMPipes-KOH,pH7.0,andcentrifugedat600gduring10min.

FluorescenceAnalysis

FluorescentBY-2tobaccocells,transfectedwithEGFPoraproteinfusedtoEGFP,wereobservedbetweenaslideandcoverslipwithaZeissLSM510confocalmicroscope(Jena,Germany).EGFPwasviewedbyexcitationat488nmwithanargonlaserusinganappropriateemission ltertocollectthegreensignalfromtheopticalsection.Fluorescentcellswerealsoobservedunderthesameconditionsafterincubationfor8hat248CwithgentleshakingintheBY-2cellculturemediumcontaining100nMleptomycinB.

Forimmuno uorescencestudies,protoplastsornucleipreparedasdescribedabovewereharvestedand xedfor15minwithgentleshakinginprotoplastornuclei-speci cmedium,respectively,containing4%glutaraldehyde.Thereafter,theywerewashedthreetimeswiththeappropriatemedium,oncewiththemediumdilutedvolumetovolumewithPBS,thenagainwithPBSand nallyresuspendedinPBSbuffer.Asampleofprotoplastsornucleiwasmountedonapoly-L-Lys–coatedcoverslip,allowedtosettlefor1hatroomtemperature,andthentreatedovernightat48Cina0.1%sodiumborohydridesolution.Protoplastsandnucleiwereincubatedfor1hinablockingsolution(5%acetylatedBSA[Aurion,Wageningen,TheNetherlands],5%normalgoatserum,and0.1%coldwater shskingelatinpreparedinPBS)andthenovernightwiththepolyclonalanti-P6antibodies.Aftersixwasheswith0.1%BSAcinPBS,protoplastsornucleiweretreatedwithgoatanti-rabbitantibodiescoupledtoAlexa488(MolecularProbes,Eugene,OR),respectively,for12h.Afterremovalofexcesssecondaryantibodiesbysixwashesin0.1%BSAcinPBS,theprotoplastsandnucleiweresubsequentlyexaminedwithaZeissLSM510confocalmicroscope.

ACKNOWLEDGMENTS

WethankMarcBergdollforthethree-dimensionalmodelingofP6andJohnStanleyforprovidinguswiththeCaMVCabb-JIgenomese-quence.WearemostgratefultoChristianeGaraudandJe

´ro meMuttererforadviceonCLSMandtoKenRichardsforcriticalreadingofthemanuscript.TheInter-InstituteConfocalMicroscopyPlatformwas

co nancedbytheRe

´gionAlsace,CentreNationaldelaRechercheScienti que,theUniversite

´LouisPasteur,andtheAssociationdelaRecherchepourleCancer.ThisworkwassupportedbytheCentre

NationaldelaRechercheScienti queandbytheUniversite

´LouisPasteurofStrasbourg.

ReceivedNovember1,2004;acceptedDecember9,2004.

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat

942ThePlantCell

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