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

CaMVP6IsaNucleocytoplasmicProtein933

Figure4.Fine-ScaleMappingoftheP6–P6InteractionDomain.

(A)SchematicrepresentationofdomainA(aminoacids1to112).TheconservedregioncanbedividedintotwosubdomainsdesignatedA1(aminoacids1to83)andA2(aminoacids84to112).Thetwoinvariantsequences(I1andI2)indomainAareindicatedbysolidlinesandthea-helixbyagraybox.aa,aminoacids.

(B)Top:thesequenceofaminoacids4to31ofP6containsfourtypicalLeuzipperheptadmotifs.Hydrophobicresiduesatheptadpositionsaanddareinbold.TheinvariantsequenceI1andthenearduplicatesequencei1areindicated.LeuresiduessubstitutedinEGFP:P6mbyaGln(atpositions14and16)andbyaHis(atposition18)arespeci ed.Bottom:acomputer-generatedmodelofaparallelcoiled-coilstructureformedbetweentheNterminioftwoP6molecules.Thesidechainsoftheresiduesinpositionsaanddareshown.

(C)GST:A,GST:A1,andGST:A2aswellasGSTandfull-lengthP6wereexpressedinE.coliandsubmittedtofarproteingelblotanalysisusing32P-labeledP6,GST:A,GST:A1,orGST:A2asprobeintheoverlay.Theradioactivecomplexesweredetectedbyautoradiographyafteranexposureof24h.Molecularmassesofmarkerproteinsareindicatedattheleft.

structureoftheNterminusofP6orthecompleteproteinremainsanopenquestion.Indeed,EGFP:P6m2couldstillgivesmallaggregatesbutlostthecapacitytoassembleintolargeperinu-clearviroplasms.

P6IsaNucleocytoplasmicShuttlingProtein

Asmentionedatthebeginning,smallaggregatesofEGFP:P6werefoundinthenucleusofBY-2cells(Figure2,panels4and5)andEGFP:P6DAwaspresentmainlyinthenucleus(Figure3B,panels1and2),althoughthecorrespondingfusionproteinshaveapproximatemolecularmassesof85and75kD,respectively,whicharehigherthanthereportedlimitforpassivediffusion

¨rlichandKutay,1999).We rstsus-acrossnuclearpores(Go

pectedthatEGFP:P6andEGFP:P6DAmightbecleavedbyacellularproteasetoproduceaspeciessmallenoughtofreelydiffusethroughthenuclearpores.SuchahypothesiswasconsistentwiththeobservationthataP6-speci cdegradationproductof42kDisfrequentlyfoundinCaMV-infectedplants(Mauleetal.,1989)andinheterologousexpressionsystems(Lehetal.,2000).However,proteingelblottingassaysperformedwithproteinsfromtransfectedBY-2cellsexpressingEGFP:P6orEGFP:P6DA,usingantibodiesraisedagainstGFP,revealedonlypolypeptidesof85and70kD,respectively(seesupplemen-taldataonline),indicatingthatnosigni cantdegradationofthefusionproteinsoccurred.Consequently,EGFP:P6andEGFP:P6DAareprobablytransportedactivelyintothenucleusoftobaccocells.

Theaforesaidobservationsraisedthequestionwhetherfull-lengthP6mightlikewiseenterthenucleusofhostplantsduringCaMVinfection.Toanswerthisquestion,wepreparedproto-plastsfromsystemicallyCaMV-infectedandhealthyturnipplantsandperformedimmunodetectionofP6usinganti-P6andsecondaryantibodiescoupledtoAlexa488.Observationofthecytosolandinparticularthenucleusby uorescentmicros-copyunderstandardconditionswasoftenhinderedbythepresenceofnumerouschloroplasts.Nevertheless,diffuse uo-rescencecouldbevisualizedwithinthenucleusbutnotthenucleolus.Generally,theprotoplastscontainednumerousP6aggregatesinproximitytothenuclearmembranesothatitwasdif culttodeterminewhethersigni cantamountsoftheimmu-nolabeledP6wereindeedwithintheinteriorofthe

nucleus.