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.