2011 The Polypyrimidine Tract-Binding Protein Affects Coronavirus RNA Accumulation Levels 

5146SOLAETAL.J.VIROL.

FIG.10.InductionofstressgranulesinSTcells.STcellswereexposedtodifferentstressconditions:(i)oxidativestressinducedby1mMsodiumarsenitefor60min,(ii)PKRactivationby4?gpoly(I:C)for6h,or(iii)ERstressinducedby2?Mthapsigarginfor1.5h.ThepresenceofPTB(red)andTIAR(green)proteinsinmock-treatedorstressedSTcellswasanalyzedbyconfocalmicroscopywithspeci?cantibodies.

theamountofcellularmRNAhnRNPUwasnotsigni?cantlytranscriptioncomplexesresponsibleforviralRNAsynthesis.increasedincytoplasmicextractsbyimmunoprecipitationwithOthercellularRNA-bindingproteins,suchasthestressgran-anti-PTBoranti-TIARantibodies(datanotshown).TheseulemarkersTIA-1andTIAR,colocalizedwithPTBincyto-resultsindicatedthatduringinfection,viralgRNAandplasmicstructures.Incontrast,SGsinducedinSTcellsbysgmRNAwereassociatedwithPTBandTIARincytoplasmicoxidativestresscontainedTIARbutdidnotincludesigni?cantRNA-proteincomplexesandprobablyregulateviralRNAandlevelsofPTB,suggestingthatPTBisaspeci?ccomponentofproteinsynthesisduringviralinfection.

cytoplasmicgranulesinducedbyTGEVinfection.Interest-ingly,virusgRNAandsgmRNAweredetectedinRNAimmu-DISCUSSION

noprecipitationassaysinassociationwithPTBandTIAR,indicatingthatPTBandTIARwerecomponentsofribo-Thispaperreportsontheidenti?cationofPTBasacellularnucleoproteincomplexesinducedbyTGEVinfectionandthatproteindirectlyorindirectlybindingtoCoVTRSsandana-thosecomplexesincludedviralRNAs.

lyzesPTBintracellularinteractionsduringTGEVinfection.DuringinfectionbyTGEV,amemberofCoVgenus?,theFunctionalexperimentsshowedthatPTBhadanegativeeffectbindingofPTBtoviralRNAsequencesinvolvedintranscrip-onviralRNAaccumulationandvirusproduction,assilencingtion,suchasTRS-LandseveralTRS-Bs,hasbeenshown.ofPTBexpressionwithspeci?csiRNAsinthehumanHuh7TheseresultsareinlinewiththosedescribedforMHV,acelllineledtoareproducibleandsigni?cantincrease(uptomemberofCoVgenus?,inwhichPTBbindstotheleader4-fold)inviralmRNA7levelsandinvirustiters(3.5-fold)inTRSandalsotothecomplementofthe3?UTR(30,44).Usingrelationtothoseforcellstransfectedwiththenegative-controlfunctionalstudies,wehaveshownthatPTB-speci?csilencingsiRNA.ApossibleeffectofPTBonviralmRNAstabilityduringTGEVinfectionledtoincreasesinRNAlevelsandcannotbeexcluded.Inaddition,therewasaninversecorrela-virusinfectivity.Theseresultsareconsistentwiththeprevi-tionbetweenPTBcytoplasmiclevelsandRNAaccumulation.ouslyreporteddecreaseinMHVRNAsynthesissubsequenttoFurthermore,PTBsilencinginhumanHEK293Tcellstrans-PTBoverexpression(15).SincePTBoverexpressioncausedanfectedwithaTGEV-derivedrepliconalsoledtoasimilarunexpectedinhibitoryeffectonviralRNAsynthesis,theseau-increaseinviralmRNA7,con?rmingthenegativeeffectofthorshypothesizedthattheexcessofPTBwoulddepleteotherPTBonviralRNAaccumulation.AfterTGEVinfection,PTBessentialfactors,indirectlyaffectingMHVreplicationandrelocalizedfromthenucleustodiscrete,temporallyregulated,transcription,butnoexperimentalevidencesupportingthiscytoplasmicgranules.ThesePTB-containinggranulesaccumu-hypothesiswasprovided.PTBhasbeenassociatedwithotherlatedinthecytoplasmofinfectedcellsatthesametimethatsingle-strandedpositive-senseRNAviruses.InhepatitisCvi-activeviralRNAsynthesisdecreased.Inlinewiththisobser-rus(HCV)andpicornaviruses,PTBfunctionsasanIRESvation,nocolocalizationbetweenPTBincytoplasmicgranulestrans-actingfactor,activatingviraltranslationinitiation(34,anddsRNAorthereplicasecomponentnsp8wasobserved,49).However,theroleofPTBinRNAreplicationofpositive-indicatingthatPTBwasnotaccumulatedinactivereplication-senseRNAvirusesisstillcontroversial.InHCV,ithasbeen

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FIG.11.PresenceofPTBincytoplasmicgranulesinducedbyTGEVinfectioninHuh7cells.Huh7cellswereinfectedwiththeTGEVPUR46-C11strain(MOI,20).ThepresenceofPTB(red)andTIAR(green)inmock-infectedandinfectedHuh7cellsat48and72hpiwasanalyzedbyconfocalmicroscopyusingspeci?cantibodies.

reportedthatPTBispartoftheRNAreplicationcomplexandparticipatesinviralRNAsynthesis(2).Incontrastwiththeseresults,ithasalsobeenreportedthatPTBpartiallyrepressesHCVreplicationandinhibitsbindingofRdRptothe3?UTR(21).Indenguevirus,PTBhasbeenshowntobeinvolvedexclusivelywiththevirusreplicationmachinery(8)andinreplicationortranslation(1).Insummary,theactivityofPTBinthereplicationofCoVsseemstofollowauniformeffectofreducingviralRNAlevels,whichisvariableinthereplicationofotherpositive-senseRNAviruses.

Inthispaper,wereportPTBredistributiontocytoplasmicstructuresduringTGEVinfectionandthatviralRNAs(gRNAandsgmRNA)wereassociatedwithPTBwithinthesenovelcytoplasmicstructurespotentiallyderivedfromSGs.Incon-FIG.12.RNAimmunoprecipitationofviralRNAswithspeci?canti-trast,nucleus-to-cytoplasmrelocalizationofPTBduringMHVPTBoranti-TIARantibodies.STcellswereeithernoninfected(?)orinfectionwasnotdescribed(15).DuringTGEVinfectionofSTinfected(?)withtheTGEVPUR46-MADstrain(MOI,10).Cytoplas-cells,PTB,TIA-1,andTIARwereidenti?edintemporallymicextractswerepreparedat16hpi.RNA-proteincomplexeswereim-munoprecipitatedintheabsenceofantibodies(NoAb)orthepresenceofregulatedcytoplasmicgranulesdirectlyorindirectlyassociatedanti-PTB(?-PTB)oranti-TIAR(?-???R)antibodiesorthenegative-withvirusRNAs.Furthermore,TGEVinfectionofhumancontrolantibodyanti-GFP(?-GFP).RNAselutedfromimmunoprecipi-Huh7cellsalsoledtotheformationofcytoplasmicgranulestatedRNA-proteincomplexeswereanalyzedbyqRT-PCRforthepres-containingPTBandTIAR,similartothosedetectedinporcineenceofassociatedviralgenomicRNA(g)orsubgenomicmRNA7(sg).TheamountofviralRNAsinimmunoprecipitatedribonucleoproteinSTcells.AlthoughSGsformedinresponsetodifferentstresscomplexeswasexpressedinrelationtoRNAlevelsintheabsenceofconditionssharemanycommonfactors,somecomponentsareantibody.Quanti?cationswerefromthreeindependentRNAimmuno-uniquetoaparticularstress(39).TIARisacommoncompo-precipitationassays.Errorbarsindicatethestandarddeviations.

nentofSGsinducedinSTcellsbyTGEVinfectionandoxi-

Downloaded from http://jvi.asm.org/ on April 12, 2015 by UNIV OF CONNECTICUT5148SOLAETAL.dativestress,whereasPTBisaccumulatedtosigni?cantlevelsonlyinTGEV-inducedSGs.TheseresultsindicatethatPTBmightbeauniquemarkerofcytoplasmicgranulesformedinSTcellsduringTGEVinfection.

PTB,TIA-1,andTIARareRNA-bindingproteinsassoci-atedwithRNAmetabolismeventsinthenucleusandthecytoplasm(9,37).Inthenucleus,PTBactsasaregulatorrepressingalternativesplicing(12,33),possiblybyinterferingwithmolecularinteractionsbetweenproteincomplexesthatmediateexonde?nition(10,33)or,alternatively,byprecludingtheassociationofsplicingfactors(17,64,68).Also,TIA-1andTIARfunctioninthenucleusassplicingregulators(27,43).Inthecytoplasm,inresponsetoenvironmentalstress,TIA-1andTIARcontributetotranslationalarrest,polysomedisassembly,andaggregationofnontranslatedpolyadenylatedmRNAstoformSGs(6).Ourpaperdescribesforthe?rsttimethatduringTGEVinfection,PTBisassociatedwithcytoplasmicstruc-tures,includingtheSGmarkersTIA-1andTIAR(38).ThesestructuresaredifferentiablefromconventionalSGsbythepresenceofPTB.Onepossibilityisthatthesestructuresmightbemodi?edstressgranulescontainingRNPcomplexesin-ducedbyTGEVinfection.ThebindingofPTBtoviralRNAmaypromoteRNA-RNAorRNA-proteininteractionsre-quiredfortheassemblyoftheseRNPcomplexes.Atthesametime,TIA-1andTIAR,de?nedtobeSGnucleators(7),mightcontributetoconcentrateviralRNAsandcellularRNA-bind-ingproteinsincytoplasmicsubdomains.Infact,TIA-1andTIARtogetherwithPTBhaverecentlybeenshowntobeinvolvedintheposttranscriptionalregulationof?-F1ATPasemRNAexpressionatthetranslationlevel,mediatedbyitsspeci?cbindingtothe3?UTRofthismRNA(32,56).IthasbeenproposedthatPTBmayenhance(56)orrepress(11)translationofspeci?ccellularmRNAs,dependingonthecel-lularcontextandonthebindingofotherregulatorytrans-actingfactors(9).

Inrecentyears,thesubcellularlocalizationofmRNAsinribonucleoproteincomplexeshasbeendemonstratedtobeapowerfulmechanismtospatiallyandtemporallyregulatevar-iousRNAprocessingeventsinthecell(29).PTBhasbeenrevealedtobeakeystructuralcomponentofRNPparticlesinvolvedinregulatingthespatiotemporalpatternofgeneex-pressionduringthedevelopmentofeukaryoticorganisms(11).Accordingly,inthecontextofTGEVinfection,PTBmight(i)inhibitviralRNAaccumulation,possiblybyinterferingwithessentiallong-distanceRNA-RNAandRNA-proteininterac-tionsestablishedthroughoutthegenomeduringdiscontinuoustranscriptionbetweenTRS-LandTRS-B,aspreviouslyde-scribedinourCoVtranscriptionmodel(63,69),and(ii)sub-sequently,atlatertimespostinfection,PTBcouldrelocalizeandconcentratetogetherwithviralRNAsatcytoplasmicRNPcomplexesdifferentfromreplication-transcriptionsites,in-cludingTIA-1andTIAR,wheretranslationorotherposttran-scriptionalprocessesproceed.Sincethesecytoplasmicstruc-turesincludednotonlySGmarkersbutalsoPTBandviralRNAs,theycouldbenamedmodi?edSGs.ThetwoproposedactivitiesforPTBinthecontextofTGEVreplication,repres-sionoftemplateswitchandrelocalizationwithviralRNAstomodi?edSGs,mimicthedualcellularfunctionofPTB(11,59)asasplicingrepressorinthenucleus(12)andaregulatorofcytoplasmicmRNAstability,localization(18),andtranslation

J.VIROL.

(11,56),respectively.Therefore,PTBmightbeoneofthefactorsinvolvedinthetransitionofviralRNAsfromtranscrip-tiontoasubsequentstageintheviralcycle.Thatbeingthecase,thecytoplasmicgranulesobservedafterTGEVinfectionmightcontributetothespatiotemporalregulationofviralRNAstoenterdifferentsteps,includingtranslationorencap-sidation.Alternatively,cytoplasmicgranulesmightfunctiontolimitviralinfectionaspartofthehostresponsebyreducingviralRNAstabilityandsubsequentlyrepressingtranslationofviralmRNAs.

Inthispaper,weprovideevidenceforchangesinducedincellRNA-bindingproteinsassociatedwithmRNAmetabo-lism.ThesechangescreatedynamiccytoplasmicdomainsinwhichTIA-1andTIARaccumulateinmodi?edcytoplasmicgranuleswithPTB,inassociationwithviralRNAs.Inaddition,wehaveshownthatviralinfectioninterferedwithP-bodyfor-mation.KnowledgeoftheinteractionsbetweenviralRNAandcellularproteinswillenableustounderstandthemolecularbasisofviralpathogenesisandtodevelopbettertherapeuticstrategiestointerferewithvirusreplication.

ACKNOWLEDGMENTS

ThisworkwassupportedbygrantsfromtheMinistryofScienceandInnovationofSpain(BIO2007-60978andPET2008-0310),theCom-munityofMadrid,Spain(S-SAL-0185-2006),theU.S.NationalInsti-tutesofHealth(ARRA-W000151845),andP?zerAnimalHealth.TheresearchleadingtotheseresultshasreceivedfundingfromtheEuro-peanCommunity’s(EC’s)SeventhFrameworkProgramme(FP7/2007-2013)undertheprojectsEMPERIE(ECgrantagreementnumber223498)andPoRRSCon(ECgrantagreementnumber245141).I.S.receivedacontractsupportedbygrantsfromtheMinistryofScienceandInnovationofSpain(BIO2007-60978).

WegratefullyacknowledgeJ.Lykke-AndersonandJ.ZiebuhrforprovidingtheindicatedantibodiesandM.Gonza′lezfortechnicalas-sistance.

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