CloningofsequencescorrespondingtoCaMVP6proteinanditstruncatedversions
GACTGGGGTTGTACTAAGGCCGCCTTAGTACAACC
catgtacaagATGGAGAACATAGAAAAACTCATAGTACAACCgcatgtacaagCTCAAGATCAGAAGTACTATTCgcatgtacaagATCCCACAAAAATCTGAGCTTAAgcatgtacaagCCAATCCCACAAAAATCTGAGgattctagaTCAATCCACTTGCTTTGAAGACgattctagaTCATGGAATTCCCTGATGAGGgattctagaTCAAGCCATCAACGGATTTGTgattctagaTCAGGAGATCTCTTTTGGGGC
gattctagaTCAAAATATGTCTTTCTCTGTGTTCTTG
CloningofthesequencecorrespondingtotheP6N-terminaldomain(aminoacids1to112)
gatccatggATGGAGAACATAGAAAAACTCctaccatggtAATTCCCTGATGAGGACGSite-directedmutagenesis
AAAATACAAATGCAAGAACACGATCTACTCTTGCATGAGGAGTTTTTGTAAGAGCAAAAATAAGCTTATATGTTCTCCATCTTGTACAGC
RestrictionSiteBamHIEcoRIEcoRIBamHIEcoRI
BsrGIBsrGIBsrGIBsrGIXbaIXbaIXbaIXbaIXbaI
NcoINcoI
Forwardprimer(þ)andreverseprimer(ÿ).Restrictionsitesintheprimersequencesarerepresentedbybold-facedlower-caseletters.PCRproductswereclonedintopGEX-2TK(A),pCK-EGFP(BandD),pETP42(C),andpETKaKS.6(D)vectors.
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).