The Open Reading Frame VI Product of Cauliflower mosaic viru(7)

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

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

936ThePlantCell

Figure7.NuclearLocalizationofP6.

Nucleifromhealthy(A)andCaMV-infectedturnipleaves(B)were xedandimmunolabeledwithrabbitanti-P6antibodiesandmouseanti-rabbitIgGcoupledtoAlexa488assecondaryantibodies(image1)andstainedwithpropidiumiodide(image2).Panel3correspondstodifferentialinterferencecontrastimagesandtheright-handimages(panel4)totheirsuperpositionwiththe uorescentandpropidium-stainedimages.Theconfocalimageswerecollectedwithafocaldepthof0.45mm(C).Aseriesofsuchopticalsectionsthroughanucleusisolatedfromaninfectedplant,anti-P6immunolabeled,andstainedwithpropidiumiodide.Bar¼5mm.

tobeessentialresiduesforthenuclearexportofP6asalreadydescribedfornucleocytoplasmicshuttlingproteinspossessingaLeu-richNESandinparticularfortheBR1proteinofSquashleafcurlvirus(WardandLazarowitz,1999).However,wecannottotallyexcludethattheinvariantsequenceI1alsohaspropertiesinvolvedintheretentionoffusionproteininthecytosol.

Therefore,thenuclearexportofP6wasfurtherinvestigated,treatingBY-2cellstransfectedwiththeaforementionedre-combinantplasmidswithleptomycinB,whichspeci callyinhib-itstheCRM-1pathwayinvolvedinthenuclearexportofmanyproteins(Fornerodetal.,1997;Kudoetal.,1998).WhenBY-2cellsexpressingEGFP:Awereincubatedwith100nMlepto-mycinB,6hafterbombardment,the uorescentproteinaccu-mulatedabundantlyinthenucleus(Figure8C,panel2),whereasno uorescencewaspresentinthiscompartmentinuntreatedcontrolcells(Figure8C,panel1),thuscon rmingthatEGFP:Amoleculesareactivelyexportedfromthenucleus.ThefusionproteinEGFP:P6formedlargeaggregatesinthecytoplasmandwasundetectableinthenucleusoftransfectedtobaccocells(Figure8C,panel3),butwhenthelatterweretreatedwithleptomycinB,itwasfoundinboththecytoplasmandinthenucleus(Figure8C,panel4).Thenuclearcompartmentcon-taineddiffuse uorescence,accompaniedasexpectedbymanysmallaggregatesbecauseP6wasabletoself-interact.ThisresultprovesthatP6isactivelytransportedbetweenthenucle-ocytoplasmiccompartments.SimilarexperimentswerealsoperformedwithpointmutatedEGFP:P6versionstodeterminethefunctionalimportanceofdifferentresiduesinthenuclearexportofP6.Asexpected,themutantEGFP:P6m1hadthesamesubcellularlocalizationinBY-2cellstreatedwithleptomycin

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