The Open Reading Frame VI Product of Cauliflower mosaic viru(6)

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.

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