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

938ThePlantCell

(datanotshown)ornot(Figure5B,panels5and6)becausemutationsoftheLeuresiduesaresuf cientpersetoimpairtheexportofthefusionprotein.The uorescenceinthecytoplasmevenafterleptomycinBtreatmentsuggeststhatnotallEGFP:P6m1moleculesenteredthenucleusbecausetheywereretainedinthecytoplasmor,alternatively,thatotherresiduesoftheI1sequencemightcontributetotheexport.IncontrastwithEGFP:P6m1,leptomycinBhadonlylittleeffectonthenuclearexportofEGFP:P6m2becausealmostno uorescencecouldbedetectedwithinthenucleus(Figure8C,panels5and6)inmostBY-2cellsobservedbyCLSM;onlyfewcellsexhibitedahighly uorescentnucleus.ThedifferenceobservedintheresponsetoleptomycinBtreatmentmightbeduetothefactthat(1)theresiduesmutatedinEGFP:P6m2areindeedessentialforexportashypothesizedpreviouslyor(2)thattheirmutationsmodifytheconformationofEGFP:P6inamannerthatinterferessomehowwiththeactivityoftheinhibitor.TheEGFP:P6m3mutantmainlyaccumulatedinthenucleoplasmwhenthecellswereincubatedinthepresenceofleptomycinB,whereasitdidnotinuntreatedcells;onlylowlevelsofEGFP:P6m3werefoundinthecytoplasmoftreatedcells(Figure8C,panels7and8).ThisresultsuggeststhattheEKImotifisnotpartoftheNES,orifitis,ithasaminorin uenceontheexportofP6bytheCRM-1pathway.

Together,thesedataleadtotheconclusionthatP6containsatitsNterminusanNESandthatitsLeuresiduesinvolvedinthenuclearexportofP6arerecognizedbytheCRM-1pathway.TheactivityofthisexportpathwaylimitstheaccumulationofP6inthenucleusthatcouldbedeleteriousfortheCaMVinfectiouscycle.DISCUSSION

DuringthecourseofaCaMVinfection,P6formselectron-denseviroplasmsthatarethoughttobevirionfactoriesthatserveasascaffoldforvirusreplicationandassembly.Inthisstudy,wehaveinvestigatedbyinvitroandinvivoexperimentalapproachesthemechanismleadingtotheformationoftheseviroplasms.OurresultsshowthatP6self-interacts,asalreadysuggested(Haasetal.,2000),andfurtherdemonstratethat,atleastinvitro,theP6–P6interactionsareexclusivelymediatedbytheN-terminalregionencompassingresidues1to83(subdomainA1).NootherP6sequencewasabletointeractinvitrowithfull-lengthP6.ThefactthatfusionofdomainAtounrelatedproteinsdrovethisinteractioninvitrodemonstratesthattheNterminusofP6issuf cientpersetopromoteself-interactionindependentlyfromneighboringsequences.OurresultsonlypartiallyagreewiththoseobtainedbyLiandLeisner(2002),whousedtheyeastdoublehybridsystemtoshowthat,inadditiontotheNterminus,threeotherdomainsofP6areabletobindfull-lengthP6,namelythemini-TAVdomain,thedownstreamadjacentsequence,andtheC-terminalregionexceptfora90-amino-acid-longse-quence.Possiblytheadditionalinteractingdomainscharacter-izedinthedoublehybridsystembindtoP6withanaf nitythatistoolowfordetectioninfarproteingelblotexperiments.In-completerenaturationofthedomainsinquestioncouldalsointerferewithactivityintheinvitroassays.Ontheotherhand,wecannottotallyexcludethepossibilitiesthatsomeoftheinter-actionsdetectedinyeastbyLiandLeisner(2002)mightbemediatedbyyeastRNAorproteins.Indeed,domainsofP6identi edbytheseauthorstobeinvolvedinP6–P6interactionshavenucleicacidbindingpropertiesand/orinteractwithcellularproteins,inparticularnuclear-localizedproteins(Parketal.,2001;Bureauetal.,2004).

TheroleofsubdomainA1intheformationofviroplasmswasinvestigatedbytransientexpressionintobaccoBY-2cellsoffull-lengthP6anddeletedversionsofP6fusedtoEGFP.Expressionoffull-lengthP6fusionproteingaverisetoviroplasmslocatedintheproximityofthenucleus,whicharestructurallysimilartothosefoundinCaMV-infectedplants.Indeed,inbothcases,theviroplasmsappearedbyconfocalmicroscopyanalysisasag-gregatesofmultiplehollowmacromolecularstructures,anditissuggestedthattheymightbeproducedstepwisebytheassem-blyofthedonut-likestructuresthatcouldbevisualizedinsometransfectedtobaccocells.SimilarhollowstructureswerealsoobservedwhenNSP2andNSP5proteins,thetwomajorviralcomponentsofdenseviroplasmsinducedbyrotaviruses,werecoexpressedinculturedhostcellsintheabsenceofotherrotaviralproteinsandofrotavirusreplication(Fabbrettietal.,1999).WeassumethatthehollowcenteroftheP6aggregatescorrespondstotheelectron-lucentholesofviroplasmsobservedbyelectronmicroscopyinCaMV-infectedcells(Xiongetal.,1982).

TheresultsobtainedbytransientexpressionoftruncatedversionsofP6clearlydemonstratethat,althoughthe83N-terminalaminoacidsofP6arenecessary,theyarenotsuf -cientfortheformationofviroplasmsandthatconsequentlyotherregion(s)ofP6areimplicatedinthisprocess.Thedomainbetweenaminoacids289and379,referredtoasD3byLiandLeisner(2002),mightbeoneofthesesequencesbecausetheseauthorshaveshownthatitplaysanimportantroleinP6self-associationwhentestedinvivousingtheyeasttwohybridsystem.ThisregionofP6isalsoengagedininteractionsneededfortranslationaltransactivation(DeTapiaetal.,1993;Parketal.,2001).Inanyevent,our ndingssuggestthattheN-terminallymediatedP6–P6interactionisaprerequisiteforfurtherinter-actionsbetweenotherP6sequencesand/orforstabilizationofmacromolecularstructuresbecauseformationofviroplasmswastotallyimpairedwhenanN-terminallytruncatedP6(P6DA)wasexpressedintobaccocells.

ComputeranalysisofsubdomainA1indicatedthatitformsanamphipathica-helixatitsNterminus(residues4to31).ThelattercontainsaLeuzippermotifthatcouldformaparallelcoiled-coilstructure(Lupas,1997),stronglysuggestingthatsuchaconfor-mationisimplicatedintheinteractionbetweenP6molecules.Thishypothesisiscon rmedbytheresultsoffarproteingelblotassaysshowingthattheintermolecularinteractionislostifkeyhydrophobicaminoacidsoftheLeuzippermotifsaresubstitutedbypolarresidues.InvolvementofsuchaninteractionintheformationofviroplasmsisevidencedbythefailureofP6carryingthesepointmutationsinthea-helixtoformaggregatesintransfectedtobaccocells.Currently,wehypothesizethatthecoiled-coilformationbetweentheNterminiofinteractingP6moleculesinducesconformationalchangesthatallowotherregionsofP6toparticipateintheaggregationprocess.Indeed,mutationsofaminoacidsthatarenotlocatedatthecoiled-coilinteractingsurfacedidnotpreventtheformationofsmall