The Open Reading Frame VI Product of Cauliflower mosaic viru(4)

Takentogether,thesedatashowthattheN-terminalregionofP6isnecessarybutapparentlynotsuf cientfortheformationofviroplasmsand,thus,thatotherregion(s)ofP6contributetothisprocess.OurfailuretodetectEGFP:AinthenucleusstronglysupportstheideathatthecytosoliclocalizationofP6isgovernedbydomainA.

TheN-TerminalP6–P6InteractionDomainIsConservedamongCaMVStrains

ComparisonoftheN-terminalsequenceofP6fromCaMVstrainCabb-BJIwithitscounterpartsfromotherCaMVstrains(CabbS,CabbS-Japan,NY8153,CM1841,W260,D/H,D4,B29,Xin/Jin,andBari)showedidentityrangingfrom83to97%.TheregioncanbedividedintotwosubdomainsthatwehavedesignatedA1(aminoacids1to83)andA2(aminoacids84to112),respectively(Figure4A).Theformeristhemostconserved(87to99%ofidentity)withtwonotableinvariantsequences:I1(aminoacids11to20)andI2(aminoacids63to83).NotethatI1alsocontainsapentapeptidemotifEKILM(residues11to15)thatisidenticalatfourof vepositionstotheupsteamsequenceEKLLM(motifi1;Figure4B,top).ThesequenceI1formspartofapredicteda-helixlocatedneartheNterminus(positions4to31)thatcontainsaseriesoffoursuccessiveheptadsequences(abcdefg),whereresiduesinpositionsaanddarehydrophobicasinLeuzippermotifs(Figure4B).Thissequenceispredictedbycomputeranalysis(Berger,1995)todimerizeviaacoiled-coilstructurewith>98%probability.SubdomainA2ismorevariable(62to93%identity)anddoesnotpossessaconservedmotifamongCaMVstrains;ithasapredictedbsheetcon guration.

Asa rststeptowardfurtherde ningtheP6–P6interactiondomain,thesequencescodingforsubdomainsA1andA2wereampli edbyPCRandclonedintothepGEX-2TKvectortoproduceGST-taggedproteins.Thecapacityofthesefusionproteinstoself-interactandtointeractwithP6orGST:Aweretestedbyfarproteingelblotexperiments.RadiolabeledP6interactedwithGST:AandGST:A1butnotwithGST:A2(Figure4C,leftpanel),showingthatsubdomainA1isresponsiblefortheinteractionbetweenP6molecules.Thisresultwascon rmedbythe ndingthat,wheneitherGST:AorGST:A1wasusedasoverlay,theyinteractedwithP6andwiththemselvesbutnotwithGST:A2,whereasthelatterwasunabletobindeithertoP6ortoanyofthefusionproteins(Figure4C,rightpanels).NotethatnoneofthefusionproteinsinteractedwithGST,excludingthepossi-bilitythattheobservedinteractionswereduetodimerizationofthetag.Takentogether,theseresultsprovideevidencethatthe83N-terminalaminoacidsencompassthedomainrequiredforP6self-interactioninvitroand,hence,probablyfortheformationofviroplasms.

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

CaMVP6IsaNucleocytoplasmicProtein931

Figure2.SubcellularLocalizationAnalysisofP6FusedtoEGFPinTobaccoBY-2CellsandofP6inProtoplastsfromCaMV-InfectedTurnipPlantsbyCLSM.

(A)Green uorescentimagesofEGFP:P6(images1and4)weretaken20haftertransfectionoftobaccocellsbybombardmentwithpCK-EGFP:P6plasmid.A0.45-mm-thickopticalsectionwassampledusingasingletrackconfocalmicroscopeandappropriate lters.Image3correspondstoanenlargementofaggregatessimilartothoseobservedinimage1.Images2and5correspondtothesuperpositionofthe uorescentimageandthecorrespondingdifferentialinterferencecontrastimage.Bars¼10mm.N,nucleus;Nu,nucleolus.

(B)ProtoplastspreparedfromCaMV-infectedturnipleaveswere xedandimmunolabeledwithrabbitanti-P6antibodiesandmouseanti-rabbitIgGcoupledtoAlexa568assecondaryantibody.Shownisthered uorescentimageofatypicalprotoplast.Theconfocalimageswerecollectedwithafocaldepthof0.45mm.Bar¼10mm.

MutationsintheN-Terminala-HelixofP6AffecttheFormationofViroplasms

InviewofthefactthatsubdomainA1istotallyconservedamongCaMVstrainsandispartoftheputativeLeuzipper–containinga-helix,additionalexperimentswereperformedtofurtherin-vestigateitsroleinviroplasmformation.Weremovedboththei1andI1sequences(aminoacidsfrompositions5to20)fromP6(Figure5A)toseeifthereductionofthesizeofthea-heliximpairstheformationofviroplasms.The uorescenceofthecorrespond-ingEGFP:P6Di1-I1mutantwasveryabundantanddiffuseinthenucleus,whereasonlylowlevelswerefoundinthecytoplasm(Figure5B,panels1and2).SimilarbehaviorwasobservedwithmutantEGFP:P6DI1,inwhichwedeletedonlytheI1sequence(Figure5B,panels3and4).TheseP6mutantsdidnotformaggregates,thusreinforcingthehypothesisthattheN-terminala-helixisrequiredforP6self-assembly.Moreover,theseresultsstronglysuggestthatthea-helixand/orspeci cresiduesofI1arealsoimplicatedinthecytoplasmiclocalizationofP6becausebothconstructions,EGFP:P6Di1-I1andEGFP:P6DI1,localizedalmosttotallyinthenucleus,incontrastwithEGFP:P6(Fig-ure2A).

Insummary,theforegoingresultsstronglysuggestthattheN-terminala-helixhasstructuralfeaturesimportantforboththeaggregationofP6anditslocalizationinthecytoplasm.Todemonstrateitsroleintheformationofviroplasm,wemutatedthreeLeuresiduesoftheI1sequenceofP6(seealsobelow).TheLeuresiduesatpositions14and16weresubstitutedbyGlnresiduesandtheLeuatposition18byaHis(Figure4B,top).Aminoacidresidues14and18correspondtoposition‘‘d’’ofthesecondheptadandtoposition‘‘a’’inthethirdheptadoftheLeuzipper.Theyarepredictedtolieonthesurfaceofthea-helixandtobeinvolvedinhydrophobicinteractionsbetweenP6mole-culesinthecoiled-coilstructure(Figure4B).Leu16,ontheotherhand,isnotpredictedtobesurface-located.

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