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

940ThePlantCell

regulatedduringtheviralcycle(inotherwords,thatitoccursonlyatspeci cstages).Thesewouldexplainthedif cultyween-counteredtodetectP6innucleifrominfectedplants.

ThestudyofthebehaviorofP6mutantsinBY-2cellsrevealedthatthenuclearexportactivityisassociatedwiththeLeu-richsequence(residues11to20)attheNterminusofP6.Itsin-volvementinnuclearexportwasdemonstratedbytheincapacityofP6toexitthenucleuswhenLeuresiduesofthesequencewerepointmutatedandbythefactthatmutateddomainAofP6accumulatesinthenucleus,incontrastwiththewild-typeform.ThesequenceEKIisnotimplicatedintheexportofP6asevidencedbytheresultsofexperimentsperformedwithlepto-mycinB,butitisanimportantdeterminantfortheformationofviroplasms.ConcerningtheinvolvementoftheotherresiduesoftheI1invariantsequence,furtherinvestigationsarenecessarytode nitivelyanswerthisquestion.Interestingly,theNESispartofthea-helixthatisinvolvedinP6self-assemblyandthisfactmightexplainwhydeletionofthe rst90nucleotidesoftheCaMVORFVIabolishessystemicinfectionandsigni cantlyreducesthereplicationofthegenomeinsinglecells(KobayashiandHohn,2003).TheoverlapbetweendomainsinvolvedinP6exportandself-assemblyalsoraisesthequestionofhowthesetwoactivitiesareregulatedduringtheviralcycle.WehypothesizethatP6proteinshuttlingbetweenthenuclearandcytoplasmiccompart-mentsprimarilyinvolvesapopulationofP6monomers(ordimers)thathaveescapedtheaggregationprocess.Recentstudieshavedemonstratedthatimportinsful lladualfunctionasanuclearimportreceptorandcytoplasmicchaperonefornuclearimported

proteins(Ja

¨keletal.,2002).SuchanantiaggregationmechanismmightalsobeinvolvedforP6molecules.Thisdoesnot,however,excludethepossibilitythatP6couldbeincorporatedintoviroplasmsaftertheirexportfromthenucleus.

ThediscoverythatP6isanucleocytoplasmicshuttleproteinopensnewprospectsforunderstandingthemechanismsbywhichthisviralproteinregulatestheCaMVinfectiouscycle.Thefunction(s)ofP6inthenucleuscanonlybeamatterforspeculationatpresent.P6mighthavearolesimilartotheRevproteinofHIV-1(PollardandMalim,1998)incontrollingexportofCaMV35SRNAanditssplicedversionsbecauseitalsohasthecapacitytobindsingle-anddouble-strandedRNA(DeTapiaetal.,1993;Cerritellietal.,1998).ThepresenceofP6inthenucleolus,whereassemblyofribosomalsubunitsoccurs,raisesthepossibilitythatP6mightinteractdirectlywithribosomesbeforetheirexporttorenderthemcompetentfortranslationoftheCaMVpolycistronicmRNA.TheribosomalproteinsL18andL24,whichinteractwiththemini-TAV(Lehetal.,2000)andRNAbindingdomains(Parketal.,2001)ofP6,respectively,couldbetargetsforP6becausetheyparticipateintheformationofthe60Ssubunitinthenucleolus(Andersenetal.,2002).OtherfunctionsmightalsobeassociatedwiththenucleocytoplasmiclocalizationofP6(i.e.,inhibitionofnonsense-mediatedmRNAdecaytopreventdegradationofthe35SRNAanditssplicedversions)(forareview,seeMaquatandCarmichael,2001).Thesehypothesesaresupportedbythe ndingthatP6nuclearexportismediatedbytheCRM-1pathway(Kudoetal.,1998),whichisknowntobespeci callyusedforexportoftheribosomalsubunitsandofsomecellularmRNAs(forareview,seeWeis,2002).

METHODS

ConstructionofRecombinantPlasmids

RecombinantplasmidswereconstructedbyinsertionofviralsequencesintothepET3aderivativespETKaKS(Lehetal.,2000),pGEX-2TK(Amersham-PharmaciaBiotech,Uppsala,Sweden),andpCK-EGFP(Clontech,PaloAlto,CA).DNAfragments ankedbyappropriatere-strictioncloningsitesweregeneratedusingPCR;theoligonucleotidesusedforPCRarelistedinTable1.

CaMVORFVIanditsderivativeswereclonedeitherintotheKpnIandSacIsitesorintotheSacIsiteofthepETKaKSplasmid.ViralDNAsequenceswereampli edfromplasmidpMD324containingtheCaMVCabb-JIgenome(DelsenyandHull,1983)usingtwoprimersbearingattheir59terminiKpnIandSacIsites,respectively,orSacIsites.TheDNAfragmentsweredigestedwiththeappropriaterestrictionenzymesandintroducedintopETKaKScleavedwithKpnIandSacIorwithSacI.Allconstructswerecon rmedtobeerrorfreebysequencing.ExpressionoftherecombinantplasmidsinEscherichiacoligeneratesfusionproteinscontainingattheirNterminusthedecapeptideMet-Arg-Arg-Ala-Ser-Val-Gly-Ser-Gly-Thr,whichcanbephosphorylatedinvitrobyaproteinkinasefrombovineheartmuscle(thephosphorylationsiteisinbold-facedtype).

TheDNAsequenceencodingtheNterminusoftheCaMVP6protein(nucleotides1to336ofORFVI)wasampli edfromthepETKaKS.6recombinantplasmidencompassingthecompleteORFVIusingprimerscarryinganNcoIrestrictionsiteattheir59end.ThePCRfragmentwasdigestedwithNcoIandclonedintopETP42(Lauberetal.,1998),whichhadbeencleavedwiththesameenzymetoproducepET-A:P42.PlasmidspGST:A(ORFVInucleotides1to336),pGST:A1(ORFVInucleotides1to249),andpGST:A2(ORFVInucleotides250to336),codingfordifferentregionsoftheP6NterminusfusedtoGST,wereobtainedbyPCRampli cationofdifferentORFVIsequenceswithprimerscontaining59terminalBamHIandEcoRIsites(Table1).Theampli edDNAfragmentsweredigestedwiththeappropriaterestrictionendonucleasesandclonedintolinearizedpGEX-2TK.

ThepCK-EGFPvectorwasusedtoconstructtherecombinantplas-midscodingforfusionproteinsbetweenEGFPandwild-typeCaMVP6orP6mutants.ThecorrespondingORFVIsequenceswereampli edbyPCRfrompMD324usingtwoprimerscarryingattheir59endsBsrGIandXbaIsites,respectively(Table1).

DeletionsandpointmutationswereintroducedinEGFP:P6andEGFP:Abysite-directedmutagenesis(Stratagene,LaJolla,CA).Therecombinantplasmidswereampli edbyPCRusingpfuTurbopoly-meraseaccordingtothemanufacturer’sinstructionsanddesignedinternaloligonucleotidesasprimers(Table1).ThemixturewasthenincubatedwithtwounitsofDpnIfor2hat378Ctodestroythetemplate.The59endsofPCRproductswerephosphorylatedbyT4polynucleotidekinaseinthepresenceof1mMATPforsubsequentligation.Error-freerecombinantplasmidswereidenti edbyDNAsequencing.

ProductionandPhosphorylationofRecombinantProteins

E.coliBL21/DE3(pLysS)strainwastransformedbyelectroporationwithpETKaKSandpGEX-2TKrecombinantplasmidscodingforfull-lengthP6orP6mutants.Expressionoftheheterologousproteinswasinducedwith1mMisopropylb-thiogalactosidefor2honcethebacterialculturehadreachedtheexponentialphase.Bacteriawerecollectedbycentrifu-gationat5000gfor5min,resuspendedinHMKbuffer(20mMTris-HCl,pH7.5,100mMNaCl,and12mMMgCl2),andlysedbysonication(threepulsesfor20sat50W).Aftercentrifugationat12,000gfor10min,thesupernatantwasdiscardedandtheinclusionbodiesresuspendedin500mLofHMKbuffer.