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.

CaMVP6IsaNucleocytoplasmicProtein935

Inconclusion,ourdataclearlysupporttheideathatP6canenterthenucleusduringviralinfectionandalsoindicatethatP6isanucleocytoplasmicshuttlingprotein.Furthermore,our ndingsalsostronglysuggestthatthesequencedownstreamofdomainAisimplicatedinthenuclearlocalizationofP6,whereastheN-terminalregionofP6mightcontainanuclearexportsignal(NES).

TheNTerminusofP6ContainsanNESThatIsRecognizedbytheCRM-1NuclearExportPathway

ThelatterhypothesisisreinforcedbythefactthatdeletionoftheconservedhydrophobicsequenceI1locatedinsubdomainA1ormutationoftheLeuresiduesatpositions14,16,and18partiallyabolishednuclearexportofP6(Figure5B,panels3to6).Moreover,thisLeu-richsequencebearssomeresemblancetotheNES(EKDTLLIDL)foundintheBR1proteinoftheSquashleafcurlvirus,ageminivirus(WardandLazarowitz,1999),andtotheNESsequenceofseveralknownrapidlyshuttlingnuclearpro-teins,suchasHIVRevprotein(forareview,seePollardandMalim,1998).

ToprovidefurtherevidencethattheaforesaidsequenceisanNES,wedeletedthesequenceI1ormutatedLeuresidues14,16,and18intheEGFP:Afusionprotein(Figure8A)asdescribedabove,andthebehaviorintobaccoBY-2cellsofthemutantswascomparedwiththatofthenonmutatedprotein.TheseexperimentswereperformedwithEGFP:Aratherthanwiththefull-lengthP6becausenuclearaccumulationofEGFP:Amutantswouldbedirectlyrelevanttotheimpairmentoftheexportprocess,whereastheaccumulationofEGFP:P6mutantsinthenucleusdoesnotpermitdiscriminationbetweenanexportdefectandanactiveimportoftheprotein.Indeed,thewild-typefusionproteinEGFP:Awasneverfoundinthenucleus,althoughitisofasizethatshouldpermitittodiffusefreelythroughthenuclearpore(Figure3B,panels3and4).

WhensequenceI1wasremovedfromdomainA,EGFP:ADI1wasequallydistributedinthecytoplasmandthenucleusexceptforthenucleolus(Figure8B,panels1and2),whereasEGFP:Alocalizedexclusivelytothecytoplasmiccompartment(Figure3B,panels3and4).ThesubcellularlocalizationinBY-2cellsofmutantEGFP:Am(Figure8B,panels3and4),inwhichthethreeLeuresiduesofI1werereplacedbypolarresidues,wassimilartothatobservedwithEGFP:ADI1(Figure8B,panels1and2);EGFP:Amwasfoundinboththecytoplasmandinthenucleusbutnotinthenucleolus.TheabsenceofbothEGFP:Amutantsinthenucleolus,incontrastwiththesituationwithEGFP:P6DI1andEGFP:P6pointmutatedversions(Figure5),mightbeduetothefactthattheN-terminalregionofP6isunabletointeractwithribosomalproteins,whereastheEGFP:P6mutantsstillcontainthecorrespondinginteractiondomains(i.e.,mini-TAVandRNAbindingdomainA)(Lehetal.,2000;Parketal.,2001;Bureauetal.,2004).

Alltheseresultssupportamodelinwhich(1)bothEGFP:AandEGFP:Amutantscanenterandexitthenucleusbydiffusion,(2)EGFP:AmoleculesbutneitherEGFP:ADI1norEGFP:Amarerapidlyexportedtothecytoplasm,and(3)thatthesequenceI1functionsasanNESbecausepointmutationsofLeuresiduesinI1impairtheexportofEGFP:A.Thus,theseLeuresidues

appear