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