The Open Reading Frame VI Product of Cauliflower mosaic viru(10)

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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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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