The Open Reading Frame VI Product of Cauliflower mosaic viru(3)

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

930ThePlantCell

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.

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