The Open Reading Frame VI Product of Cauliflower mosaic viru(13)

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

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.