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.,