The Open Reading Frame VI Product of Cauliflower mosaic viru(5)

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

932ThePlantCell

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

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