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
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
CaMVP6IsaNucleocytoplasmicProtein939
aggregates;however,theyimpairedassemblyoftheseaggre-gatesintoviroplasms.Itisevidentthatafurtherunderstandingofthemechanismofviroplasmformationwillalsorequirecharac-terizationoftheotherdomainsofP6implicatedinthisprocessaswellaspossiblecellularstructuresand/orfactorsthatmightbeinvolved,suchasendomembranesand/orthemicrotubularnetwork.Nevertheless,nohost-speci cfactorsseemtobeneededbecauseviroplasmsthatformedintobaccocells,anon-hostforCaMV,aresimilartothosefoundinhostcells.Asnotedearlier,membranecomponentsmayfunctionintheearlystepsofP6self-assembly,characterizedbyformationofsmallaggregatesasthelatterdisappearedupontreatmentoftobaccocellswithnonionicdetergent,whereastheviroplasmslocatedatthenuclearperipherywereunaffected(datanotshown).Furtherinvestigationswillberequiredtodeterminewhetherbindingtoendomembranesisessentialfortheself-assemblyofP6andwhetheritshydrophobicNterminus,whichpresentsfeaturesofapeptidesignal(BlobelandDobberstein,1975),playsarole.Becauseviroplasmformationdoesnotvisuallyperturbthemicrotubuleortheactin lamentnetworksinBY-2cells,itwillbeofparticularinteresttodeterminewhetherviroplasmformationinvolvesthecytoskeleton,asdescribedinanimalcellsforaggresomesandfortheviralfactoriesinducedbylargecytoplasmicDNAvirusessuchasAfricanswinefevervirus(Kopito,2000;Heathetal.,2001).Johnstonetal.(1998)pro-posedamodelinwhichsmallaggregatesaredeliveredbyretrogradetransportalongmicrotubulestotheperipheryofthenucleuswheretheyareassembledintolargestructures.
TransientexpressionofEGFP-taggedP6ledtotheunex-pecteddiscoverythatP6,considereduntilnowasacytoplasmicprotein,isactuallyanucleocytoplasmicshuttlingprotein.Thepresenceofthisviralproteinwithinthenucleuswascon rmedbyitsimmunodetectioninnucleipreparedfromCaMV-infectedturnipleavesandCLSM-generatedopticalserialsectionsthroughtheorganelles.Ourresults,obtainedwithtransfectedtobaccocells,indicatethatonlyasmallfractionofP6,probablymoleculesthatarenotengagedintheaggregationprocess,entersthenucleus.Indeed,EGFP:P6mainlyformedinclusionbodiesinthecytoplasmandwasalmostundetectableinthenucleusoftobaccocells.OnlyinhibitionoftheexportprocessbyleptomycinBsystematicallypermittedobservationofdiffuseEGFP:P6andsmallaggregateswithinthenucleus.OurresultsalsoindicatethattheexportofP6probablyoccursveryrapidlyininfectedcells,sothatonlylowamountsarepresentinthenucleusatanytime.ThestrongP6nuclearexportactivityprobablypreventsaccumulationofP6withinthenucleus,whichcouldbedeleteriousforCaMVinfectivity.Inaddition,itismorethanlikelythatthenucleocytoplasmictransportis nely
Table1.OligonucleotidesUsedasPrimerstoGeneratethePCRProductsClonedintopGEX-2TK,pCK-EGFP,pETP42,andpETKaKS.6VectorsNameA
ABam(þ)AEco(ÿ)A1Eco(ÿ)A2Bam(þ)A2Eco(ÿ)B
Bsrmut(þ)Bsrmut(ÿ)P6Bsr(þ)PBsr(þ)P6DABsr(þ)QBsr(þ)P6Xba(ÿ)AXba(ÿ)A1Xba(ÿ)BXba(ÿ)QXba(ÿ)C
ANco(þ)ANco(ÿ)D
NESmut(þ)DNES(ÿ)NES(þ)Di1(ÿ)
Sequence
CloningofsequencescorrespondingtoCaMVP6truncatedversionsgccggatccATGGAGAACGAAAAACTCgatgaattcTCATGGAATTCCCTGATGAGGcacgaatccCTAAGCCATCAACGGATTTGggcggatccTCCAATATCTTGTCAAAAGATcacgaattcCTATTCTGCTCTGAGAGGAGC