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
ThePlantCell,Vol.17,927–943,March2005,ª2005AmericanSocietyofPlantBiologists
TheOpenReadingFrameVIProductofCauli owermosaicvirusIsaNucleocytoplasmicProtein:ItsNTerminusMediatesItsNuclearExportandFormationofElectron-DenseViroplasms
`leGeldreich,aMarinaBureau,aLaurenceDupuis,aVe´roniqueLeh,a,2GuillaumeVetter,aMurielHaas,a,1Ange
KappeiKobayashi,bThomasHohn,bLyubovRyabova,aPierreYot,aandMarioKellera,3
´culairedesPlantes,Unite´PropredeRecherche,CentreNationaldelaRecherchedeBiologieMole
´LouisPasteur,67084StrasbourgCedex,FranceScienti que2357,Universite
bFriedrichMiescherInstitute,CH-4002Basel,Switzerland
aInstitut
TheCauli owermosaicvirus(CaMV)openreadingframeVIproduct(P6)isessentialfortheviralinfectioncycle.ItcontrolstranslationreinitiationoftheviralpolycistronicRNAsandformscytoplasmicinclusionbodies(viroplasms)wherevirusreplicationandassemblyoccur.Inthisstudy,themechanisminvolvedinviroplasmformationwasinvestigatedbyinvitroandinvivoexperiments.FarproteingelblotassaysusingacollectionofP6deletionmutantsdemonstratedthattheN-terminala-helixofP6mediatesinteractionbetweenP6molecules.Transientexpressionintobacco(Nicotianatabacum)BY-2cellsoffull-lengthP6andP6mutantsfusedtoenhancedgreen uorescentproteinrevealedthatviroplasmsareformedattheperipheryofthenucleusandthattheN-terminaldomainofP6isanimportantdeterminantinthisprocess.Finally,thisstudyledtotheunexpected ndingthatP6isanucleocytoplasmicshuttleproteinandthatitsnuclearexportismediatedbyaLeu-richsequencethatispartofthea-helixdomainimplicatedinviroplasmformation.ThediscoverythatP6canlocalizetothenucleusopensnewprospectsforunderstandingyetunknownrolesofthisviralproteininthecourseoftheCaMVinfectioncycle.
INTRODUCTION
Cauli owermosaicvirus(CaMV)isthetypememberoftheCaulimovirusgenusoftheCaulimoviridaefamily.Itsdouble-strandedcircularDNA(;8kb)isreplicatedviaanRNAin-termediatebyavirus-encodedreversetranscriptase,andthevirusisclassi edinthepararetrovirussupergroup,towhichanimalvirusesoftheHepadnaviridaefamilyalsobelong(forareview,seeHaasetal.,2002).TheCaMVgenomepossessessixmajoropenreadingframes(ORFsItoVI),whicharealllocatedonthesameDNAstrand.ManyfunctionsofthecorrespondinggeneproductsP1toP6havebeenelucidated,butthemecha-
´PropredeRecherche,CentreNationaldelaaddress:Unite
´partementRe´cepteursetProte´inesRechercheScienti que9050,De
´rieuredeBiotechnologiedeStrasbourg,Membranaires,EcoleSupe
´bastientBrant,67412IllkirchCedex,France.boulevardSe
2Currentaddress:LaboratoiredeDynamique,EvolutionetExpression
´nomesdeMicroorganismes,FormationdeRechercheendesGe
´Evolution2326,CentreNationaldelaRechercheScienti que,Universite
LouisPasteur,28rueGoethe,67084StrasbourgCedex,France.
3Towhomcorrespondenceshouldbeaddressed.E-mailmario.keller@ibmp-ulp.u-strasbg.fr;fax33-3-88-61-44-42.
Theauthorresponsiblefordistributionofmaterialsintegraltothe ndingspresentedinthisarticleinaccordancewiththepolicydescribedintheInstructionsforAuthors()is:MarioKeller(mario.keller@ibmp-ulp.u-strasbg.fr).versioncontainsWeb-onlydata.
Article,publicationdate,/cgi/doi/10.1105/tpc.104.029017.
1Current
nismsbywhichtheyoperateduringviralinfectionarenotyetfullyunderstood.
TheviralDNAistranscribedbythecellularRNApolymeraseIIintotwomajorcappedandpolyadenylatedRNAs,amonocis-tronic19SmRNAandapregenomic35SRNAthatservesasatemplatebothforreversetranscriptionandfortranslationintoP1toP5.The35SRNAundergoesalternativesplicingeventsleadingtofourmRNAsinwhichORFIandpartofORFIIaredeleted(Kiss-Laszloetal.,1995).Currently,themechanismregulatingthenuclearexportof35SRNAanditssplicedformsisunknown.TheseRNAsaretranslatedbythecellularmachineryfollowingtwounconventionalstrategies,ribosomalshuntandtermination–reinitiation(forareview,seeRyabovaetal.,2002).TheCaMVP6protein(62kD),whichisexpressedspeci callyfromthe19SRNA,isamultifunctionalproteinthatrepresentsakeycomponentintheCaMVinfectiouscycle.P6isthemajordeterminantofhostspeci cityandin uencessymptomseverity(Daubertetal.,1984;DaubertandRouth,1990;Agamaetal.,2002).InoculationofcruciferousandsolanaceousplantspecieswithchimericCaMVgenomesbearingORFVIderivedfromdifferentCaMVisolatesshowedthattheN-terminalregionofP6isresponsibleforhostspeci city(Daubertetal.,1984;Schoelzetal.,1986).TransgenicArabidopsisthalianaplantsexpressingP6displaydiseasesymptomswhoseseverityisrelatedtotheexpressionlevelofthetransgene(Zijlstraetal.,1996).Compar-isonofthecellularmRNAcontentofORFVI-transgenicandcontrolArabidopsisplantsrevealedthatORFVIexpressiondownregulatesorupregulatesseveralhostgenes(Gerietal.,
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