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

JOURNALOFVIROLOGY,May2011,p.5136–5149Vol.85,No.10

0022-538X/11/$12.00doi:10.1128/JVI.00195-11

Copyright?2011,AmericanSocietyforMicrobiology.AllRightsReserved.

ThePolypyrimidineTract-BindingProteinAffectsCoronavirusRNA

AccumulationLevelsandRelocalizesViralRNAstoNovel

CytoplasmicDomainsDifferentfromReplication-TranscriptionSites?IsabelSola,CarmenGala′n,?PedroA.Mateos-Go′mez,LorenaPalacio,SoniaZu′n?iga,

JazminaL.Cruz,FernandoAlmaza′n,andLuisEnjuanes*

DepartmentofMolecularandCellBiology,CentroNacionaldeBiotecnología,CSIC,Darwin3,Cantoblanco,28049Madrid,Spain

Received28January2011/Accepted2March2011

Thecoronavirus(CoV)discontinuoustranscriptionmechanismisdrivenbylong-distanceRNA-RNAinter-actionsbetweentranscription-regulatingsequences(TRSs)locatedatthe5?terminalleader(TRS-L)andalso

precedingeachmRNA-codingsequence(TRS-B).ThecontributionofhostcellproteinstoCoVtranscriptionneedsadditionalinformation.Polypyrimidinetract-bindingprotein(PTB)wasreproduciblyidenti?edinassociationwithpositive-senseRNAsoftransmissiblegastroenteritiscoronavirus(TGEV)TRS-LandTRS-Bbyaf?nitychromatographyandmassspectrometry.AtemporalregulationofPTBcytoplasmiclevelswasobservedduringinfection,withasigni?cantincreasefrom7to16hpostinfectionbeinginverselyassociatedwithadecreaseinviralreplicationandtranscription.SilencingtheexpressionofPTBwithsmallinterferingRNAintwocelllines(Huh7andHEK293T)ledtoasigni?cantincreaseofupto4-foldinmRNAlevelsandvirustiter,indicatinganegativeeffectofPTBonCoVRNAaccumulation.DuringCoVinfection,PTBrelocalizedfromthenucleustonovelcytoplasmicstructuresdifferentfromreplication-transcriptionsitesinwhichstressgranulemarkersT-cellintracellularantigen-1(TIA-1)andTIA-1-relatedprotein(TIAR)colo-calized.PTBwasdetectedinthesemodi?edstressgranulesinTGEV-infectedswinetestiscellsbutnotinstressgranulesinducedbyoxidativestress.Furthermore,viralgenomicandsubgenomicRNAsweredetectedinassociationwithPTBandTIAR.Thesecytoplasmicribonucleoproteincomplexesmightbeinvolvedinpost-transcriptionalregulationofvirusgeneexpression.

Transmissiblegastroenteritisvirus(TGEV)isamemberofboseADP1?-phosphatase,andasecondRNA-dependenttheCoronaviridaefamily,includedintheNidoviralesorder(25,RNApolymeraseresidinginnonstructuralprotein8(nsp8)26).Coronaviruses(CoVs)arethecausativeagentsofavariety(31).Inadditiontothereplicasecomponents,theviralnucleo-ofrespiratoryandentericdiseasesinhumansandanimalsproteinhasbeenshowntoplayamajorroleinCoVRNA(22,53).Theemergenceofsevereacuterespiratorysyn-synthesis(3,60,70).ThestructuresoftheCoVgenomicanddromecoronavirus(SARS-CoV)revealedthepotentialhighsubgenomicRNAsresemblethestructureofmostcellularpathogenicityofCoVsforhumansbyinfecting8,000peoplemRNAs,containingacapstructureatthe5?end,apoly(A)tailandkillingabout10%ofthem(52).Commonancestorsofatthe3?end,and5?and3?untranslatedregions(UTRs).CoVCoVshavebeenidenti?edinbatsdistributedworldwide,suggestingthattheymayrepresentanaturalreservoirfromgeneexpressiondependsonadiscontinuoustranscriptionwhichvirusesmaybereintroducedintothehumanpopula-processleadingtoacollectionofsubgenomicmRNAstion(20,42,46,54,55).

(sgmRNAs),consistingofthe5?terminalleadersequence(L)CoVshavethelargestknownRNAgenome,consistingofajoinedtodistantgenomicsequences.Thiscomplexprocessissingle-strandedpositive-senseRNAofabout30kbinlengthassociatedwithtranscription-regulatingsequences(TRSs),lo-(19,25,50).TheCoVreplicasegene,whichoccupiesthe5?catedatthe3?endoftheleader(TRS-L)andprecedingeachtwo-thirdsofthegenome,isextremelycomplex,andbesidesgene(bodyTRSorTRS-B).TRSsincludetheconservedcoretheRNA-dependentRNApolymerase(RdRp)andhelicasesequence(CS)(5?-CUAAAC-3?),identicalinallTGEVgenes,activities,itencodesotherenzymeslessfrequentorexclusiveandthe5?and3??ankingsequences(5?TRSand3?TRS,amongRNAviruses(50,62,67),suchasanendoribonuclease,respectively)(24).

a3?-5?exoribonuclease,a2?-O-ribosemethyltransferase,ari-InagreementwiththeproposedworkingmodelforCoVtranscription(63,69),TRS-Bwouldactasanattenuationanddissociationsignalforthetranscriptioncomplexduringthe*Correspondingauthor.Mailingaddress:DepartmentofMolecularsynthesisoftheminus-strandRNA.ThistranscriptionstepandCellBiology,CentroNacionaldeBiotecnología,CSIC,Darwin3,wouldpromoteatemplateswitchofthenascentRNA,com-Cantoblanco,28049Madrid,Spain.Phone:34915854555.Fax:3491plementarytothecodingsequences,tothegenome5?leader5854506.E-mail:L.Enjuanes@cnb.csic.es.

?Presentaddress:Max-PlanckInstituteofImmunobiology,Depart-region.Then,thesynthesisofminus-strandsubgenomicRNAmentofEpigenetics,LaboratoryJenuwein,Stu¨beweg51,D-79108(sgRNA)wouldresume,addingacopyoftheleader.TheFreiburg,resultingchimericsgRNAsofminussenseserveastemplates?Germany.Publishedaheadofprinton16March2011.

toyieldsgmRNAsthatshareboth5?and3?terminalsequences

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Downloaded from http://jvi.asm.org/ on April 12, 2015 by UNIV OF CONNECTICUTVOL.85,2011withthegenomeRNA.PreviousstudiesontheTGEVtran-scriptionmechanismhaveshownthatcomplementaritybe-tweenTRS-LandcomplementofTRS-B(cTRS-B)inthenascentRNAisadeterminantfactorduringtemplateswitch(63,69).Presumably,hostcellproteinsalsocontributetotran-scriptionalregulationbyRNA-proteinandprotein-proteinin-teractionsinvolvingTRSs(24,50).

Todate,limitedinformationoncellularproteinsinvolvedinCoVtranscriptionisavailable.Theheterogeneousnuclearri-bonucleoproteins(hnRNPs)A1andQwereidenti?edthroughtheirinteractionwithTRSsofthemousehepatitisvirus(MHV),amemberofCoVgenus?.TheseproteinswerecharacterizedaspossiblepositiveregulatorsforviralRNAsynthesis(16,45).Thepolypyrimidinetract-bindingprotein(PTB)wasalsodescribedtobindtheMHVleaderTRS(30).However,analysisoftheroleofPTBinMHVreplicationandtranscriptiondidnotleadtoclearconclusions(15).

PTB,alsoknownashnRNPI,isamemberofthehnRNPfamilyofRNA-bindingproteins,whichregulatedifferentas-pectsofRNAmetabolismbothinthenucleusandinthecytoplasmofeukaryoticcells(59).Inthenucleus,PTBactsasapre-mRNAsplicingrepressorassociatedwithtissue-speci?cexons(12).IthasbeenproposedthatPTBinterfereswithmolecularinteractionsacrosstheexonbetweenproteincom-plexesthatmediateexonde?nition(33)or,alternatively,byprecludingtheassociationofsplicingfactorsrequiredforexonRNAremoval(64).Inthecytoplasm,PTBisinvolvedintheregulationofcap-independenttranslationofviralandcellularmRNAsdrivenbyinternalribosomeentrysite(IRES)(61),mRNAlocation(47),andstability(41).

Wehaverecentlyreportedontheinteractionofcellularproteinswiththe5?and3?UTRsoftheTGEVRNAgenome,amemberofCoVgenus?.ThebindingofPTBtothe5?endoftheviralgenomewasshown(28).Inthestudydescribedinthisreport,PTBinteractionwithTGEVTRSshasbeenshownbyRNAaf?nitychromatographyandmassspectrometryanal-ysis.ThefunctionalrelevanceofPTBonTGEVtranscriptionwasanalyzedbysmallinterferingRNA(siRNA)approachesinhumanHuh7cellsinfectedwithTGEVandinHEK293TcellstransfectedwithaTGEV-derivedreplicon.Asigni?cantin-creaseofupto4-foldinmRNAlevelsandvirustiterwasobservedaftersilencingoftheexpressionofPTB,suggestinganegativeeffectofPTBinTGEVinfection.InTGEV-infectedcells,PTBlocalizedtonoveldiscretecytoplasmicgranulesatthetimethatRNAsynthesisceased.Neitherdouble-strandedRNA(dsRNA),intermediatesofviralRNAsynthesis,norcomponentsoftheviralreplication-transcriptioncomplexweredetectedinthesecytoplasmicstructures.However,cellularRNA-bindingproteinssuchasT-cellintracellularantigen-1(TIA-1)andtheTIA-1-relatedprotein(TIAR)wereidenti?edinPTB-containinggranules.PTBwasnotdetectedinTIAR-containingstressgranules(SGs)inducedinswinetestis(ST)cellsbyoxidativestress,suggestingthatPTBmightbeaspeci?ccomponentofcytoplasmicgranulesinducedbyTGEVinfec-tion.Interestingly,viralgenomicRNA(gRNA)andsgmRNAweredetectedinassociationwithPTBandTIAR.ThesedataindicatethatviralRNAsandcellularproteinssuchasPTB,TIA-1,andTIARformcytoplasmicribonucleoproteincom-plexesthataremostlikelyinvolvedintheposttranscriptionalregulationofvirusgeneexpressionduringinfection.

PTBAFFECTSCORONAVIRUSRNALEVELS5137

TABLE1.OligonucleotidesusedforPCRampli?cations

Oligonucleotide

Oligonucleotidesequence(5?33?)aT7-TRSL-EcoRI-VS...................CCGGAATTCTAATACGACTCACTATAGG

GTTCTTTTACTTTAACTAGCCTTGTG

TRSL-HindIII-DraI-RS......................CCCAAGCTTTTTAAACTGAATGGAAAT

AATC

cTRSL-EcoRI-SacI-VS.......................CCGGAATTCGAGCTCTTCTTTTACTT

TAAC

T3-cTRSL-HindIII-RS.................CCCAAGCTTAATTAACCCTCACTAAAGG

GGAATGGAAATAATCAACGCTTG

aRestrictionendonucleasesitesusedforcloningareinitalics.Transcriptionpromotersareinboldface.

MATERIALSANDMETHODS

Cellsandviruses.STcells(51)weregrowninDulbeccomodi?edEaglemedium(DMEM)supplementedwith10ttalbovineserum(FBS).Humanliver-derivedHuh7cellswerekindlyprovidedbyR.Bartenschlager(UniversityofHeidelberg,Heidelberg,Germany)andweregrowninDMEMsupplementedwith10%heat-inactivatedFBS.HumanHEK293TcellsweregrowninDMEMsupplementedwith5?S.TheTGEVPUR46-MADstrain(58)wasusedtoinfectSTcells,andtheTGEVPUR46-C11strain(57)wasusedtoinfectHuh7cells.VirustitrationwasperformedonSTcellmonolayersaspreviouslyde-scribed(35).Foroxidativestressinduction,STcellswereexposedto1and3mMsodiumarsenite(Sigma)incompletemediumfor60and90minat37°C.Forendoplasmicreticulum(ER)stressinduction,STcellswereexposedto1and2?Mthapsigargin(Sigma)incompletemediumfor1.5,8,and16hat37°C.ForactivationofRNA-activatedproteinkinase(PKR),STcellsweretransfectedwith2and4?gofpoly(I:C)(Sigma)byareversetransfectionprotocolwithLipofectamine2000(Invitrogen),followingthemanufacturer’sinstructions.Stressgranuleformationwasanalyzedbyimmuno?uorescenceat2,6,and16hposttransfection.

DNAconstructs.TogenerateaDNAtemplatefortheinvitrotranscriptionofanRNAincludingTRS-L,nucleotides(nt)39to159oftheTGEVgenomewereampli?edbyPCRfromplasmidpBAC-TGEV-?Cla(4)withtheoligonucleo-tidesT7-TRSL-EcoRI-VS(whereVSindicatesvirussenseandwhichincludestheT7promoter)andTRSL-HindIII-DraI-RS(whereRSindicatesreversesense)(Table1).Fortheinvitrotranscriptionofaminus-senseRNAincludingthecomplementofTRS-L(cTRS-L),TGEVgenoment38to154wereampli?edfromthesameplasmidbyPCRwiththeoligonucleotidescTRSL-EcoRI-SacI-VSandT3-cTRSL-HindIII-RS,whichincludestheT3promoter(Table1).BothPCRampliconsweredigestedwithEcoRIandHindIIIandclonedintothesamerestrictionsitesofthevectorpSL-1190togenerateplasmidspSL-T7-TRSLandpSL-T3-cTRSL,respectively.pSL-T7-TRSLandpSL-T3-cTRSLwerelinearizedwithDraIandSacI,respectively.LinearizedplasmidpSL-T3-cTRSL-SacIwastreatedwithT4DNApolymerase(NewEnglandBioLabs)togeneratebluntends,followingthemanufacturer’sconditions.TheDNAtemplateswerepuri?edwithQIAquickreagent(Qiagen)andthenusedfortheinvitrotranscriptionreactions.PCRswereperformedwithplatinumPfxDNApolymerase(Invitro-gen),followingthemanufacturer’srecommendedconditions.AllcloningstepswerecheckedbysequencingthePCR-ampli?edfragmentsandcloningjunctions.Invitrotranscription.InvitrotranscriptionreactionstogenerateTRS-L-121(TGEVnt39to159)andcTRS-L-117(thecomplementofTGEVnt38to154)RNAswereperformedfrom1.5?goflinearizedpSL-T7-TRSLandpSL-T3-cTRSLtemplatesusingaMAXIscriptT7/T3transcriptionkit(Ambion),accord-ingtothemanufacturer’sinstructions.Biotin-14-CTP(Invitrogen)wasaddedata?nalconcentrationof0.16mMina1:6.25ratiotounlabeledCTP.Thetranscriptionreactionmixtureswereincubatedfor2hat37°Candtreatedwith10unitsofDNaseIfor15minat37°C.Theresultingtranscriptswerepuri?edwithanRNeasykit(Qiagen),followingtheRNAcleanupprotocol,analyzedbydenaturingelectrophoresisin2%(wt/vol)agarose–2.2Mformaldehydegels,andquanti?edspectrophotometrically.

Cellextracts.Forproteomicsanalysis,Huh7cellsweregrownin15-cm-diam-eterdishestocon?uenceandinfectedatamultiplicityofinfection(MOI)of5withTGEVPUR46-C11.Afteranadsorptionperiodof1h,theinoculummediumwasreplacedbyfreshmediumandthecellextractswerepreparedat

Downloaded from http://jvi.asm.org/ on April 12, 2015 by UNIV OF CONNECTICUT5138SOLAETAL.

TABLE2.Biotin-labeledRNAsequencesusedinaf?nity

chromatographyexperiments

NameTRSSequence(5?33?)aPolarity

Size(nt)

TRS-L-30TRS-LCACCAACUCGAACUAAACGAAAUAUUUGUC?30cTRS-L-16TRS-LAUUUCGUUUAGUUCGA?16TRS-S2-30TRS-S2GAAACCUUCCUUCUAAACUAUAGUAGUAGG?30cTRS-S2-16TRS-S2CUAUAGUUUAGAAGGA?TRS-N-30TRS-NCAUAUGGUAUAACUAAACUUCUAAAUGGCC?1630cTRS-N-30

TRS-N

GCCAUUUAGAAGGUUUAGUUAUACCAUAUG

?

30

aTheconservedCS(inboldface)isthecentralmotifofRNAoligonucleotides.

72hpostinfection(hpi).Thecellswerethenwashedwithcoldphosphate-bufferedsaline(PBS),scrapedofftheplates,centrifugedat1,000?gfor5minat4°C,andstoredat?80°C.Cytoplasmicextractswerepreparedfrominfectedcellsaspreviouslydescribed(28).Extractswerestoredin10%glycerolat?80°C.TotalproteinconcentrationwasdeterminedwithaCoomassieplusproteinassay(Pierce).

5?BiotinylatedRNAoligonucleotides.5?BiotinylatedRNAoligonucleotides16or30ntlong,includingsequencesofTGEVTRS-LandTRS-Bwithpositiveornegativepolarity(Table2)andtheCSasthecentralmotif,werepurchasedfromCureVac(Tu¨bingen,Germany).

RNAaf?nitychromatography.Cellextracts(250?g)werediluted1:3inbinding-washing(BW)buffer(50mMHEPES,pH7.9,150mMKCl,5%glyc-erol,0.01%NP-40)andpreclearedthreetimeswith20?lofstreptavidin-coupledDynabeads(M-80;Dynal)for4hat4°C.Invitro-transcribedRNAs(3?g)or5?biotinylatedRNAoligonucleotides(400pmol)weredilutedin20?lofRNA-bindingbuffer(5mMTrisHCl,pH7.5,0.5mMEDTA,1MNaCl)andincubatedwith20?loffreshstreptavidin-coupledDynabeadsfor30minatroomtemperature.TheimmobilizedRNAwaswashedthreetimeswith200?lofBWbufferandthenincubatedwiththepreclearedproteinextractovernight.TheRNA-proteincomplexeswerewashedthreetimeswith200?lofBWbuffer.RNA-interactingproteinswereelutedwith12?lofKCl,2M,dialyzedagainstwateronnitrocellulosemembranes(VSWP01300;Millipore),resuspendedinNuPagesamplebuffer(Invitrogen),andanalyzedbydenaturingelectrophoresisusingNuPAGE4to12%bis-Trisgelsandmorpholinepropanesulfonicacid(MOPS)-SDSrunningbuffer(Invitrogen).ThegelswerewashedthreetimesindeionizedwaterandstainedwithCoomassieblueSimplyBlueSafestain(Invit-rogen),andtheproteinbandsofinterestwereexcisedfromthegelsfortheiridenti?cationbymassspectrometry.

Identi?cationofproteinsbymassspectrometry.Proteinsamplesfromexcisedbandswereanalyzedbymatrix-assistedlaserdesorptionionization–timeof?ight(MALDITOF)massspectrometryinanABI4800MALDITOF/TOFmassspectrometer(AppliedBiosystems),followedbycomparativedataanalysiswiththeNCBIhumanproteinnonredundantdatabaseusingtheMascotprogram,aspreviouslydescribed(28).

siRNAtransfection.HumanHuh7cellsweretransfectedfollowingareversetransfectionprotocol.Brie?y,foreachwellofa24-wellplate,5?104cellswereincubatedinsuspensionwith50nMPTBP1-speci?csiRNA(sensesequence5?GGAUUCAAGUUCUUCCAGAtt3?andantisensesequence5?UCUGGAAGAACUUGAAUCCtt3?[lowercaseindicatesnucleotideprotrudingatthe3?ends];catalogno.12337;Ambion)and2?lofsiPORTamine(Ambion)dilutedin50?lofOpti-MEMIreducedserummedium(GibcoBRL-Invitrogen),followingthemanufacturer’sinstructions.Asanegativecontrol,anirrelevantvalidatedsiRNA(sequencenotavailable;siRNAsequenceidenti?er4390843;Ambion)wastransfected.CellswereplatedontoeachwellusingDMEMwith10%heat-inactivatedFBS,incubatedat37°Cfor48h,andtheninfectedwithTGEVPUR46-C11atanMOIof5.At24,48,and72hpi,totalRNA,protein,andcellsupernatantswerecollectedforfurtheranalysis.HEK293Tcellsweretransfectedaspreviouslydescribed(28).Brie?y,cellsgrownto60%con?uenceweretransfectedwith100nMthesamePTBP1-speci?csiRNAandRNAiMax(Invitrogen),accordingtothemanufacturer’sspeci?cations.Cellswereincubatedat37°Cfor24handthentrypsinizedandseededin24-wellplatesatacon?uenceof2?105cellsperwell.Thecellswereretransfectedwith50nMsiRNAsat48hafterthe?rsttransfectionandincubatedfor5hat37°C.Then,

J.VIROL.

thetransfectionmediumwasdiscardedandthecellsweretransfectedwith800ngoftheTGEV-derivedrepliconREP2andLipofectamine2000(Invitrogen)aspreviouslydescribed(3).TotalRNAwascollectedforfurtheranalysisat19,28,and47hafterthereplicontransfection(72,92,and100hafterthe?rsttransfectionofsiRNA,respectively).

AnalysisofcellulargeneexpressionandviralRNAlevels.CellulargeneexpressionandviralRNAlevelswerequanti?edbyquantitativereal-timereversetranscription-PCR(qRT-PCR).TotalRNAwaspreparedwithanRNeasykit(Qiagen),accordingtothemanufacturer’sinstructions.cDNAwassynthesizedwithrandomhexamersfrom100ngoftotalRNAusingahigh-capacitycDNAtranscriptionkit(AppliedBiosystems).CellulargeneexpressionwasanalyzedusingahumanPTB-speci?cTaqMangeneexpressionassay(Hs00259176_m1PTBP1;AppliedBiosystems).ToanalyzeviralRNAlevels,acustomTaqManassay(AppliedBiosystems)speci?cforTGEVmRNA7wasused(28).DatawereacquiredwithanABIPrism7000sequencedetectionsystem(AppliedBiosystems)andanalyzedwithABIPrism7000SDS,version1.0,software.Relativegeneexpressionwasreferredtothatforcellstreatedwithavalidatednegative-controlsiRNA(Ambion)foreachtimepoint.Thedatarepresenttheaveragesofbiologicaltriplicates.

Westernblotanalysis.Celllysateswereanalyzedbydenaturingelectropho-resisinNuPAGE4to12%bis-TrisgelswithMOPS-SDSrunningbuffer(Invit-rogen).Proteinsweretransferredtoanitrocellulosemembrane(Hybond-Cextranitrocellulose;AmershamBiosciences)withaBio-RadMiniproteanIIelectro-blottingapparatusat100Vfor1hinbis-Tristransferbuffer(25mMbis-Tris,25mMbicine,1mMEDTA)containing20%methanol.Membraneswereblockedfor1hwith5%driedskimmilkinTris-bufferedsaline(20mMTris-HCl,pH7.5,150mMNaCl)andthenprobedwithantibodiesspeci?cforPTB(mousemono-clonalantibody[MAb]fromhybridomaBB7;ATCC),TGEVnucleoprotein(N;mouseMAb3DC10)(48),and?-actin(mouseMAbab8226;Abcam).Boundantibodiesweredetectedwithhorseradishperoxidase-conjugatedrabbitanti-mousesecondaryantibodyandtheImmobilonWesternchemiluminescentsub-strate(Millipore),followingthemanufacturer’srecommendations.Densitomet-ricanalysisofPTBand?-actinbandsfromatleastfourdifferentexperimentswasperformedusingQuantityOne,version4.5.1,software(Bio-Rad).

Immuno?uorescence.TGEV-infected(MOI,10)ornoninfectedSTcellswere?xedwith100%chilledmethanolfor10minatroomtemperature,washedthreetimesinPBS,andincubatedwithblockingbuffer(PBScontaining10%bovineserumalbumin)for1hatroomtemperature.Primaryantibodies(anti-PTBMAbfromhybridomaBB7fromATCC;anti-TIARandanti-TIA-1fromSantaCruzBiotechnology;anti-Dcp1akindlyprovidedbyJ.Lykke-Anderson,UniversityofColorado;anti-HCoV229Ensp8kindlyprovidedbyJ.Ziebuhr,GiessenUni-versity,Giessen,Germany;anti-N-proteinMAb3DC10[48];anti-dsRNAMAbfromEnglish&Scienti?cConsulting,Hungary)weredilutedinPBS–5%bovineserumalbumin(1:1,000foranti-TIAR,anti-TIA-1,andanti-Dcp1a;1:300foranti-nsp8;1:200foranti-dsRNAandanti-PTB;1:100foranti-N)andincubatedwithcellsatroomtemperaturefor1h.Cellswerethenwashedfourtimesfor10mineachtimewithPBSandincubatedfor1hatroomtemperaturewithsecondaryantibodiesconjugatedtoAlexaFluor488orAlexaFluor594diluted1:500inPBS–5%bovineserumalbumin.Fordouble-labelingexperimentswithMAbsagainstPTBanddsRNA,af?nity-puri?edanti-PTBMAbBB7wasdirectlylabeledwithZenonlabelingreagent(MolecularProbes,Invitrogen)followingthemanufacturer’sinstructions.Cellswere?rstincubatedwiththeprimaryMAbIgG2aanti-dsRNAandsecondaryantibodyconjugatedtoAlexaFluor594.Then,cellswereincubatedwiththecomplexesformedbyMAbBB7boundtogoatanti-mouseIgG2bFabfragmentsconjugatedtoAlexaFluor488.TopreventtransferoftheZenonlabelbetweenantibodies,a?xationwith4%formaldehydesolutioninPBSfor15minatroomtemperaturewasperformed.NuclearDNAwasvisualizedwith4?,6-diamidino-2-phenylindole(DAPI).CoverslipsweremountedinProlongGoldantifadereagent(Invitrogen)andanalyzedwithaconfocal?uorescencemicroscope(TCSSP5;Leica).Foreachexperimentalseries,imageswereacquiredwiththesameinstrumentsettingsandanalyzedwithLeicasoftware.

RNAIP.IsolationofPTBandTIAR-associatedRNAsundernativeconditionswasperformedbyimmunoprecipitation(IP)usinganti-PTBMAbBB7andgoatanti-TIARantibody,respectively.CytoplasmicextractswerepreparedfromSTcellsuninfectedorinfectedwithTGEVPUR46-MADatanMOIof10.STcellsgrownin15-cm-diameterdishestocon?uencewerewashedwithcoldPBS,scrapedofftheplates,andcentrifugedat2,000?gfor2minat4°C,andthecellpelletswereresuspendedin1mlcoldPBS.Then,thecellsuspensionwasmixedwith1mllysisbuffer(150mMNaCl,3mMMgCl2,20mMTris-HCl,pH7.5,1%NP-40,proteaseinhibitorcocktail[Roche],1.6U/?lRNasinRNaseinhibitor[Promega])bygentlepipetting,incubatedat4°Cfor10min,andcentrifugedat3,000?gfor2minat4°C.Thesupernatant,correspondingtothecytosolic

Downloaded from http://jvi.asm.org/ on April 12, 2015 by UNIV OF CONNECTICUTVOL.85,2011FIG.1.RNAaf?nitychromatographyassaysforisolationofpro-teinsinteractingwithTGEVTRS.(A)SchemeoftheRNAaf?nitychromatographyassay.(B)ProteinsfromthecytoplasmicextractsofinfectedHuh7cellswerepulleddown,separatedbySDS-PAGE,andstainedwithCoomassieblue.BandsdetectedinthepresenceofTRSRNAsandabsentinsampleswithoutRNAwereexcisedandanalyzedbymassspectrometry(MS).ArrowsindicateisoformsofPTB.Molec-ularsizemarkersareshown.TRS-L,TRS-L121-ntRNA;cTRS-L,cTRS-L117-ntRNAcomplementarytoleaderTRS;TRS-N,30-ntRNAincludingtheN-geneTRS;cTRS-N,30-ntRNAcomplementarytoTRS-N;TRS-S2,30-ntRNAincludingTRS-S2withinSgene;cTRS-S2,16-ntRNAcomplementarytoTRS-S2.(C)Schemeshowingthreeiso-formsofPTBgeneratedbyalternativesplicingofexon9.Completeskippingofexon9producesPTB1.Inclusionofexon9fromtwoalterna-tivesplicesitesproducesPTB2andPTB4,whichhaveanextra19-and26-amino-acidinsert,respectively,betweenexons8and10.

fraction,wascollectedandpreclearedbeforeRNAIPonproteinA/Gplates(proteinA/GplateIPkit;Pierce),followingthemanufacturer’sinstructions.Puri?edanti-PTB,anti-TIAR,oranti-green?uorescentprotein(anti-GFP;BoehringerMannheim)antibodieswere?rstboundtoA/Gplatesdiluted(20?g/?l)inIPbuffer(PBS,1%TritonX-100),andthencytoplasmicextractswereaddedtotheproteinA/G-antibodyplates.ImmunoprecipitatedRNA-proteincomplexeswereelutedaccordingtothemanufacturer’sinstructions.RNAwasisolatedbyanRNeasykit(Qiagen)andsubjectedtoqRT-PCRforthedetectionofPTB-,TIAR-,orGFP-associatedviralorcellularRNAs.ViralgRNAwasdetectedwithacustomTaqManassay(forwardprimer,5?-TTTAACTAGCCTTGTGCTAGATTTTGTC-3?;reverseprimer,5?-AAATAATCAACGCTTGTCCTCTATGA-3?;minorgroovebinderDNAprobe,5?-CAACTCGAACTAAACGAAAT-3?).sgmRNA7wasquanti?edasdescribedabove.

RESULTS

InteractionofPTBwithTGEVtranscription-regulatingse-quences.SinceTRSsarespeci?callyassociatedwithtranscrip-tion,theywereselectedtoisolateTRS-interactingcellularproteinspotentiallyinvolvedinviraltranscriptionbyRNAaf?nitychromatography.Biotin-labeledRNAs,includingviralTRS(Table2),wereusedtocaptureproteinsfromcytoplasmicextractsofCoV-infectedhumanHuh7cells(MOI,5).Ahu-

PTBAFFECTSCORONAVIRUSRNALEVELS5139

FIG.2.SilencingofPTBexpressioninTGEV-infectedcells.Hu-manHuh7cellsweretransfectedwithsiRNAsandinfectedwiththeTGEVPUR46-C11strainat48hposttransfection.TotalRNAandproteinextractswerecollectedat24,48,and72hpi(72,96,and120hposttransfection,respectively)toanalyzePTBsilencing.(A)AnalysisofPTBsilencingatthemRNAlevel.TheamountofPTBmRNAincellstransfectedwithPTB-speci?csiRNAwasquanti?edbyqRT-PCRandexpressedasapercentageofmRNAreferencelevelsincellstransfectedwithavalidatednegative-controlsiRNA(siRNAC?)foreachtimepostinfection.(B)AnalysisofPTBsilencingattheproteinlevelbyimmunoblottingwithanti-PTBantibody.Ponceaustainingwasusedasaloadingcontrol.

mancelllinewasselectedforproteomicanalysistoimproveproteinidenti?cation,sincehumansequencesarebetterrep-resentedinpublicdatabasesthanthosefromporcinespecies(28).RNA-proteincomplexeswereimmobilizedonstreptavi-din-coupledparamagneticbeadsandelutedproteinswerere-solvedbySDS-PAGE.BandsdetectedinthepresenceofTRSRNAsandabsentinsampleswithoutRNAwereexcised,di-gestedwithtrypsin,andsubjectedtoMALDI-TOFmassspec-trometryanalysis(Fig.1A).PTBwasreproduciblyassociatedwithpositive-senseRNAscontainingTRS-Lsequencesof30nt(TRS-L-30)or121nt(TRS-L-121),aswellas30-ntTRS-Bsequences(TRS-S2-30andTRS-N-30).Incontrast,PTBwasnotdetectedwhentheminus-strandRNAcomplementarytotheseTRSs(cTRS-L-117,cTRS-S2-30,cTRS-N-30)wasused.Twobandswithapparentmolecularmassesof57and59kDa,compatiblewithisoformsPTB1andPTB2/4,respectively,gen-eratedbyalternativesplicingofPTBmRNA(66)wereiden-ti?edwithsigni?cantscores(P?0.05)andsequencecoverage(47to88%)(Fig.1BandC).

EffectofPTBexpressionsilencingonTGEVRNAlevelsandinfectiousvirusproduction.Toanalyzethefunctionalrele-vanceofPTBonTGEVtranscriptionandinfectiousvirusproduction,itsexpressionwassilencedwithspeci?csiRNAsinthehumancelllineHuh7,whichissusceptibletoTGEVstrainPUR46-C11infection.Additionally,theeffectofPTBonTGEVRNAlevelswasevaluatedinhumanHEK293TcellstransfectedwithaTGEV-derivedreplicon(3,28).Ahumancelllinewasselectedforfunctionalassaysbecausegenesilenc-ingandgeneexpressionreagentswerenotavailablefortheporcinePTBgene,whereastheywereavailableforthehumangene.Furthermore,thedesignofporcine-speci?ccustom

Downloaded from http://jvi.asm.org/ on April 12, 2015 by UNIV OF CONNECTICUT5140SOLAETAL.FIG.3.EffectofsilencingPTBexpressiononTGEV-infectedhumanHuh7cellsandHEK293TcellstransfectedwithaTGEV-derivedrepli-con.Toanalyzetheviralphenotype,totalRNAandsupernatantswerecollectedattheindicatedtimesfromhumanHuh7cellswhichhadbeentransfectedwithsiRNAsandinfectedwithTGEVPUR46-C11andhu-manHEK293TcellstransfectedwithsiRNAsandsubsequentlywiththeTGEV-derivedreplicon.(A)Quanti?cationbyqRT-PCRofviralmRNA7accumulationincellstransfectedwithPTB-speci?csiRNAcomparedtoreferencelevelsfromcellstransfectedwithavalidatednegative-controlsiRNA(C?)ateachtimepostinfection.(B)VirusproductioninHuh7cellswasquanti?edbytitrationofthesupernatantsonSTcells.(C)EffectofsilencingPTBexpressiononhumanHEK293TcellstransfectedwithaTGEV-derivedreplicon.PTBmRNAandviralmRNA7accumulationlevelswerequanti?edbyqRT-PCRat19,28,and47hafterthereplicontransfection(hpt;72,92,and100hafterthe?rsttransfectionofsiRNA,respectively)andexpressedaspercentagesofmRNAreferencelevelsincellstransfectedwithavalidatednegative-controlsiRNA(C?)foreachtimepostinfection.Theexperimentwasperformedthreetimes,andthedatarepresenttheaveragesoftriplicates.Standarddeviationsareindi-catedaserrorbars.

siRNAsisrestrictedsinceinformationonporcinegenomicsequencesinpublicdatabasesisverylimitedandcurrentlyavailablecomputeralgorithmshavebeendevelopedbytakingasareferenceonlyhuman,mouse,andratsequences.Syn-

J.VIROL.

FIG.4.KineticanalysisofPTBcytoplasmiclevelsandviralmark-ersinTGEV-infectedSTcells.TotalRNAandcytoplasmicproteinextractswerecollectedfromSTcellsinfectedwiththeTGEVPUR46-MADstrainattheindicatedhpi.(A)WesternblotdetectionofPTBincytoplasmicextractsfrominfectedSTcellsatdifferenttimespostin-fection.Thesmallestbanddetectedwithanti-PTBMAbinporcineSTcellscorrespondstoanonspeci?cproduct.TGEVNwasdetectedasacontrolofviralinfection.Thesmallerbandcorrespondstoaproductofcaspase-mediated?given-ActininwaskDa.usedproteolysisofTGEVnucleocapsidprotein(23).(B)asQuanti?cationaloadingcontrol.ofviralProteinmRNAmolecular7andcytoplasmicmassesarePTBlevelsinTGEV-infectedSTcells.ViralmRNA7levelsatdiffer-enttimespostinfectionweredeterminedbyqRT-PCRandexpressedasrelativeunitsinreferencetotheamountat0hpi.CytoplasmicPTBlevelswerequanti?edbydensitometryofthePTB2/4band(upperband)andnormalizedagainsttheamountof?-actin.DensitometricanalysisofPTBand?-actinbandsfromatleastfourdifferentexperi-mentswasperformed,withsimilarresults.

theticsiRNAsweretransfectedintoHuh7cellsbyreversetransfection.After48h,thecellswereinfectedwiththeTGEVstrainPUR46-C11atanMOIof5.SilencingexperimentswereoptimizedtoselecttheminimalconcentrationofsiRNAandtransfectionreagentprovidingmaximumgenesilencingandminimumcytotoxicity.FrompreviousPTB-silencingexperi-ments(datanotshown),oneoutofthreespeci?csiRNAsprovidingthehighestsilencingef?ciency(?90%)waschosenforfurtheranalysis.Moreover,asinglesiRNAtransfectionwassuf?cienttoachievesustainedPTBsilencingatboththemRNAandproteinlevelsatthetimesofthephenotypicanal-ysis.PTBsilencingdidnothaveasigni?cantimpactoncellviability,ascon?rmedbytheobservedgrowthkineticsoftrans-fectedcells.Sampleswerecollectedforanalysisat24,48,and72hpi(i.e.,72,96,and120haftertransfectionofthesiRNAs,respectively).PTBmRNAlevelsshowedasigni?cantreduc-tion(90to95%)inPTB-silencedcells,inrelationtothecellstransfectedwithavalidatednegative-controlsiRNA,asdeter-minedbyqRT-PCRwithspeci?cTaqMangeneexpression

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