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Protease 2A induces stress granule formation during coxsackievirus B3 and enterovirus 71 infections

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Title:
Protease 2A induces stress granule formation during coxsackievirus B3 and enterovirus 71 infections
Creator:
Wu, Shuo
Wang, Yan
Lin, Lexun
Si, Xiaoning
Wang, Tianying
Zhong, Xiaoyan
Tong, Lei
Publisher:
BioMed Central (Virology Journal)
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English

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Infections ( jstor )
Plasmids ( jstor )
Hela cells ( jstor )

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Abstract:
Background: Stress granules (SGs) are granular aggregates in the cytoplasm that are formed under a variety of stress situations including viral infection. Previous studies indicate that poliovirus, a member of Picornaviridae, can induce SG formation. However, the exact mechanism by which the picornaviruses induce SG formation is unknown. Method: The localization of SG markers in cells infected with coxsackievirus B3 (CVB3) or enterovirus 71 (EV71) and in cells expressing each viral protein was determined via immunofluorescence assays or plasmid transfection. Eight plasmids expressing mutants of the 2A protease (2Apro) of CVB3 were generated using a site-directed mutagenesis strategy. The cleavage efficiencies of eIF4G by CVB3 2Apro and its mutants were determined via western blotting assays. Results: In this study, we found that CVB3 infection induced SG formation, as evidenced by the co-localization of some accepted SG markers in viral infection-induced granules. Furthermore, we identified that 2Apro of CVB3 was the key viral component that triggered SG formation. A 2Apro mutant with the G122E mutation, which exhibited very low cleavage efficiency toward eIF4G, significantly attenuated its capacity for SG induction, indicating that the protease activity was required for 2Apro to initiate SG formation. Finally, we observed that SGs also formed in EV71-infected cells. Expression of EV71 2Apro alone was also sufficient to cause SG formation. Conclusion: Both CVB3 and EV71 infections can induce SG formation, and 2Apro plays a crucial role in the induction of SG formation during these infections. This finding may help us to better understand how picornaviruses initiate the SG response. Keywords: Stress granule, 2A protease, Coxsackievirus B, Enterovirus 71
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Wu et al. Virology Journal 2014, 11:192 http://www.virologyj.com/content/11/1/192; Pages 1-10
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doi:10.1186/s12985-014-0192-1 Cite this article as: Wu et al.: Protease 2A induces stress granule formation during coxsackievirus B3 and enterovirus 71 infections. Virology Journal 2014 11:192.

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University of Florida
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University of Florida
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© 2014 Wu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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RESEARCHOpenAccessProtease2Ainducesstressgranuleformation duringcoxsackievirusB3andenterovirus71 infectionsShuoWu1,YanWang1,LexunLin1,XiaoningSi1,TianyingWang1,XiaoyanZhong1,LeiTong1,YingLuan1, YangChen1,XiaoyuLi3,FengminZhang1,WenranZhao2*andZhaohuaZhong1*AbstractBackground: Stressgranules(SGs)aregranularaggregatesinthecytoplasmthatareformedunderavarietyof stresssituationsincludingviralinfection.Previousstudiesindicatethatpoliovirus,amemberof Picornaviridae ,can induceSGformation.However,theexactmechanismbywhichthepicornavirusesinduceSGformationisunknown. Method: ThelocalizationofSGmarkersincellsinfectedwithcoxsackievirusB3(CVB3)orenterovirus71(EV71)and incellsexpressingeachviralproteinwasdeterminedviaimmunofluorescenceassaysorplasmidtransfection.Eight plasmidsexpressingmutantsofthe2Aprotease(2Apro)ofCVB3weregeneratedusingasite-directedmutagenesis strategy.ThecleavageefficienciesofeIF4GbyCVB32Aproanditsmutantsweredeterminedviawesternblotting assays. Results: Inthisstudy,wefoundthatCVB3infectioninducedSGformation,asevidencedbytheco-localizationof someacceptedSGmarkersinviralinfection-inducedgranules.Furthermore,weidentifiedthat2AproofCVB3was thekeyviralcomponentthattriggeredSGformation.A2ApromutantwiththeG122Emutation,whichexhibited verylowcleavageefficiencytowardeIF4G,significantlyattenuateditscapacityforSGinduction,indicatingthatthe proteaseactivitywasrequiredfor2AprotoinitiateSGformation.Finally,weobservedthatSGsalsoformedin EV71-infectedcells.ExpressionofEV712AproalonewasalsosufficienttocauseSGformation. Conclusion: BothCVB3andEV71infectionscaninduceSGformation,and2Aproplaysacrucialroleintheinductionof SGformationduringtheseinfections.ThisfindingmayhelpustobetterunderstandhowpicornavirusesinitiatetheSG response. Keywords: Stressgranule,2Aprotease,CoxsackievirusB,Enterovirus71BackgroundStressgranules(SGs)aregranularaggregatesformedin thecytoplasmofeukaryoticcellsexposedtoavarietyof environmentalstressconditions,e.g.,heatshock,UVirradiation,hypoxia,endoplasmicreticulumstress,andviral infection[1,2].SGstypicallycontaintranslationallysilent mRNAs,40Sribosomalsubunits,eukaryoticinitiation factors(eIFs)suchaseIF4E,eIF4G,eIF4A,eIF4B,eIF3, andeIF2,andRNA-bindingproteins(RBPs),including poly(A)-bindingprotein(PABP1),theembryoniclethal abnormalvision(ELAV)Huprotein(HuR),polysomal ribonuclease1(PMR-1),tristetraprolin(TTP),T-cellrestrictedintracellularantigen1(TIA1),TIA-1-related protein(TIAR),fragileXmentalretardationprotein (FMRP),andRas-GapSH3-bindingprotein(G3BP1)[1,2]. Amongtheserecruitedproteins,HuR,TIA1,andG3BP1 canactasmarkersofSGs[1-4]. Numerousviruseshavebeenshowntointeractwith SGswithdifferenteffects[5-7].Somevirusesinducestable SGformationduringinfection,e.g.,respiratorysyncytial virus(RSV).Someviruses,e.g.,mammalianorthoreoviruses(MRV),SemlikiForestviruses(SFV),andhepatitis Cviruses(HCV)havebeenshowntoinduceSG *Correspondence: zhaowr@ems.hrbmu.edu.cn ; zhongzh@hrbmu.edu.cn2DepartmentofCellBiology,HarbinMedicalUniversity,Harbin150081,China1DepartmentofMicrobiology,HarbinMedicalUniversity,Harbin150081, China Fulllistofauthorinformationisavailableattheendofthearticle ©2014Wuetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle, unlessotherwisestated.Wu etal.VirologyJournal 2014, 11 :192 http://www.virologyj.com/content/11/1/192

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formationbuttodisassemblethesegranulesasinfection proceeds.Poliovirus(PV)inducesSGformationearlyduringinfectionandlaterinhibitsSGsthroughtheviral 3Cpro-mediatedcleavageofG3BP1.Meanwhile,TIA1containingfocidevoidofotherSG-definingcomponents, suchasinitiationfactorsandmostmRNAswereobserved latepost-infection.Someviruses,e.g.,WestNilevirus (WNV),denguevirus(DV),rhesusrotavirus(RRV),type 1humanT-cellleukemiaviruses(HTLV-1),andhuman immunodeficiencyviruses(HIV)werefoundtobeableto suppressSGformation[5-7].Cardioviruseswithmutant leader(L)proteins,suchasTheiler ’ smurineencephalomyelitisvirus(TMEV),encephalomyocarditisvirus(EMCV) andSaffoldvirus(SAFV)induceSGformationthroughout theinfection,whileexpressionoftheLproteinduringinfectionefficientlyblocksSGformation.Similarly,influenzaAvirus(IAV)infectionfailstoinduceSGsunless viruseswithNS1mutationsareused[5-9].Insomecases, SGsplayanimportantroleinhostantiviraldefense,e.g., IAV,MRVandvacciniavirus(VV).However,SGsinduced byRSVcanpromotevirusreplication[5-7]. GroupBcoxsackieviruses(CVBs)arethemajorpathogensofhumanviralmyocarditisanddilatedcardiomyopathy[10,11].Enterovirus71(EV71)isthemajorpathogen ofhumanhand,foot,andmouthdisease(HFMD)[12]. BothCVBsandEV71belongtotheenterovirusgenusof the Picornaviridae family.Thepicornavirusgenomeisa ~7.0-kb-to-8.5-kbsingle-strandedpositive-senseRNA (+ssRNA)thatiscomposedofasingleopenreadingframe (ORF)andtwountranslatedregions(UTRs)atits5 and 3 flanks.ThegenomecanactasanmRNAencodinga polyproteinthatisproteolyticallyprocessedbyviralproteinasesintostructuralandnonstructuralproteins[13]. Picornavirusesinducemultiplealterationsinhostcellsto facilitateitsreplication.Suppressingcellularbiosynthesis byviralproteinases2A(2Apro)and3C(3Cpro)isoneof themostnotablealterations[13-17].Uponthecleavageof eIF4G,eIF5B,andPABPby3Cproand2Apro,picornavirusescanshutoffcap-dependenttranslationandterminatecellularbiosynthesis[13-17].Inaddition,throughthe cleavageofnuclearporecomplexproteins(Nups),2AprocanalterRNAandproteintraffickingbetweenthenucleus andcytoplasm[13].Arecentreportshowsthat2Aproof PV,amemberof Picornaviridae ,cancauseadramatic nuclear-cytoplasmre-localizationofSRp20,acellularsplicingfactorthatisalsodefinedasanIREStrans-actingfactor(ITAF)[18]. RecentstudiesindicatethatPVinducesuniqueSGsin infectedcells[19-21].ThePV-inducedSGscontaincertaincomponentsthatdonotlocalizetoSGsinducedby oxidativestress,e.g.,Sam68andSRp20[20,22].However, theexactmechanismbywhichthepicornavirusinduces SGformationisunknown.Inthisstudy,weconfirmed thatCVBtype3(CVB3)andEV71alsoinduceSG formation.Wefoundthat2Aproplaysacrucialrolein SGinductionbecause2Aproaloneissufficienttotrigger SGformation.Ourfindingsmayhelpustobetter understandthemechanismbywhichpicornavirusesinitiateSGformation.ResultsCVB3infectioninducesSGformationTofacilitateobservingSGformationduringCVB3infection,weconstructedaHeLacellline(HeLaEGFP-TIA1), constitutivelyexpressingEGFP-TIA1.SGformationwas firstdeterminedbyobservingtheexpressionand localizationofTIA1andHuR.HeLaEGFP-TIA1cellswere infectedwithCVB3(MOI=10)ortreatedwith0.5mM NaArsfor30min.Inthemock-treatedcells,EGFP-TIA1 wasdistributedinboththecytoplasmandnucleus.Inthe Ars-treatedandCVB3-infectedcells,EGFP-TIA1was granularlydistributedinthecytoplasm.Thegranules begantoemergeinthecytoplasmoftheCVB3-infected cellsat3hpost-infection(p.i.)(Figure1). Usingananti-HuRoranti-G3BP1antibody,wefound thatHuRwaspredominantlylocalizedinthenucleusof themock-infectedcells,butitre-localizedtothecytoplasmicgranulesafterbothArstreatmentandCVB3infection (Figure1A).G3BP1wasdistributedevenlyinthecytoplasmofthemock-infectedcellsbutformedgranulesin theCVB3-infectedcellsat3hp.i.buttheG3BP1-positive granulesdispersedat6hp.i.(Figure1B).EGFP-TIA1and HuRwereco-localizedinthegranulesinArs-treatedand CVB3-infectedcells(Figure1A).G3BP1wasalsocolocalizedwithEGFP-TIA1inthegranulesofArs-treated (datanotshown)andCVB3-infectedcells(Figure1B). Furthermore,inHeLacellsco-transfectedwith pEGFP-eIF4GandpmCherry-HuR,bothmCherry-HuR andEGFP-eIF4Gre-locatedfromthenucleustothecytoplasmicgranulesafterCVB3infection(MOI=10)for3h (Figure1C).WealsonoticedthattheSGscontainedHuR butnoeIF4Gat6hp.i.(Figure1C).ThesedatademonstratethatCVB3inducedtypicalSGs.2AproisthekeyproteinthattriggersSGformationToidentifytheviralcomponentthattriggeredSGformationduringCVB3infection,HeLacellswerecotransfectedwithpmCherry-HuRandoneofthenine plasmidsexpressingEGFP-taggedviralproteins(VP1, VP4-3,2Apro,2B,2C,3A,3B,3Cpro,and3D).WeobservedthatmCherry-HuRwastranslocatedfromthenucleustothecytoplasmicgranulesincellstransfectedwith pEGFP-2Aafter24hpost-transfection(Figure2A).We observedasimilarresultinVerocells(Additionalfile1: FigureS1).ThenuclearlocalizationofmCherry-HuRwas notaffectedincellsexpressingEGFP,VP1,VP4-3,2B,2C, 3A,3B,3Cpro,or3D(Figure2A).Quantificationofthese experimentsindicatedthat93%(±2%[standarddeviation])Wu etal.VirologyJournal 2014, 11 :192 Page2of10 http://www.virologyj.com/content/11/1/192

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ofCVB32A pro -expressingcellsexhibitedSGs,while thepercentagesofSGsinothergroupsofcellsdidnot exceed5%. BymonitoringtheendogenousHuRdistributionusing theanti-HuRantibody,asimilarresultwasobservedin cellsonlytransfectedwiththeviralprotein-expressing Figure1 CVB3infectioninducesSGformation.(A) HeLa EGFP-TIA1 cellsweremockinfected,exposedto0.5mMNaArsfor30min,orinfected withCVB3(MOI=10)for3h.ThecellswerethenfixedandstainedforHuR. (B) HeLa EGFP-TIA1 cellswereinfectedwithCVB3asdiscribedin (A) .Thecells werethenfixedandstainedforG3BP1. (C) HeLacells,co-transfectedwithpEGFP-eIF4GandpmC herry-HuR,wereinfectedwithCVB3asdiscribedin (A) .Thecellswerethenfixedandimaged.NucleiwereidentifiedbyHoechst33342 staining.Cellswereexaminedusingafluorescencemicroscopy(×400). Wu etal.VirologyJournal 2014, 11 :192 Page3of10 http://www.virologyj.com/content/11/1/192

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plasmids(Figure2B).Identically,G3BP1detectionshowed thatG3BP1wasdistributedincytoplasmicgranulesonly inthecellsexpressing2A pro (Figure2C).Theseresults implythat2A pro isthekeyviralcomponentthattriggers SGformationduringCVB3infection. Theproteaseactivityisnecessaryfor2A pro totriggerSG formation BecausetheaminoacidsequencesofCVB32A pro andPV 2A pro werehighlyhomologous(Figure3),eightEGFPtaggedCVB32A pro mutantsincludingD39E,L40F,S67F, Y89L,Y90L,V120M,G122E,andD136N(markedwith asterisksinFigure3)weregeneratedaccordingtothePV 2A pro variantsthatexhibiteddifferentcleavageactivitytowardeIF4G(Additionalfile1:TableS4).Theirprotease activities,asindicatedbytheirefficiencyincleavingeIF4G [23-25],wereevaluated.Table1showstheresultsoftheir assessmentofeIF4Gcleavageactivity.Incellsexpressing thesemutants,only2A G122E showedattenuatedprotease activitycomparedwiththeprototype(Figure4Cand Table1).ThemutantsincludingL40F,S67F,V120M, G122E,andD136Nbehavedasexpected[24,25],whilethe Figure2 CVB32A pro isidentifiedastheviralcomponentthatinducesSGformationinHeLacells.(A) HeLacellswereco-transfectedwith thepmCherry-HuRplasmidandoneofthefollowingplasmids:pEGFP-2A,pEGFP-2B,pEGFP-2C,pEGFP-3A,pEGFP-3B,pEGFP-3C,pEGFP-3D, pEGFP-VP1,orpEGFP-VP4-3.At24hpost-transfection,thecellswereimaged.ThequantificationofSGformationforeachgroupwasdetermined byanalyzingthepercentageofEGFP-expressingcellsthatcontainedthreeormoregranules.Atleast100cellswerecountedforeachexperiment. Theaverageofthreeindependentexperimentsisindicatedonthelowerpartofeachpanel. (B) HeLacellsweretransfectedwithpEGFP-C1, pEGFP-2A,orpEGFP-3C.Thecellswerethenfixedat24hpost-transfectionandstainedforHuRviaananti-HuRrabbitpolyclonalantibodyanda CF555-labeledgoatanti-rabbitsecondaryantibody. (C) HeLacellsweretreatedasdiscribedin (B) andthenstainedforG3BP1viaananti-G3BP1 mousemonoclonalantibodyandaCF555-labeledgoatanti-mousesecondaryantibody.Hoechst33342wasusedtocounterstainthenuclei.The SGsweredeterminedbyobservingthegranulardistributionofmCherry-HuR (A) ,HuR (B) ,andG3BP1 (C) inthecytoplasmunderafluorescence microscope(×400).Thecytoplasmicgranulesareindicatedbyarrowheads. Wu etal.VirologyJournal 2014, 11 :192 Page4of10 http://www.virologyj.com/content/11/1/192

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mutantsD39E,Y89L,andY90Ldidnotappeartobehave asexpected[23]. Todeterminewhethertheproteinaseactivityof2A pro is requiredforSGformation,HeLacellswereco-transfected withpmCherry-HuRandeachofthemutant-expressing plasmids.WefoundthatonlyafewSGswereformedin cellsexpressing2A G122E .Incontrast,therewereabundant SGsformedinthecellsexpressingother2A pro mutants (Figure4A).Quantitativeanalysisofthesedatarevealed thatonly7%(±3%)ofEGFP-2A G122E -expressingcellscontainedSGsandover90%ofcellsexpressingtheother2A mutantsremainedpositiveforSGs.Similarresultswere observedforG3BP1astheSGmarker(Figure4B).Therefore,weconcludedthattheproteaseactivitywasrequired for2A pro toinduceSGs. EV71andits2A pro alsoinduceSGformation WenextexaminedwhetherSGsformedincellsinfected withEV71.HeLa EGFP-TIA1 cellsweremock-infectedorinfectedwithEV71atanMOIof10.Intheinfectedcells, bothEGFP-TIA1andHuRunderwentredistributionand co-localizedtothecytoplasmicgranules(Figure5A).At 3hp.i.,approximately34%oftheinfectedcellsshowed granules.ThegranulesalsocontainedG3BP1andeIF4G (datanotshown),indicatingthatEV71infectionalso inducedSGformation.Similartotheexperimentsdescribedintheprevioussection,HeLacellswerealso co-transfectedwithpmCherry-HuRandpEGFP-C1or pEGFP-EV712A.Wefoundthat91%(±4%)ofEV71 2A pro -expressedcellswerepositiveforSGs(Figure5B). AsimilarresultwasobtainedintheinfectedVerocells (Additionalfile1:FigureS1).Thus,EV712A pro can functioninSGformation. Discussion Theinteractionbetweenvirusesandhostcellsiscritical forunderstandingthepathogenesisofviralinfections. SGformationisoneoftheremarkablecellularevents whencellssenseanunfavorablestimulation[1,2].MultiplevirusesinteractwithSGsindifferentways[5-7].It hasbeenreportedthatPVcaninduceSGformation. CVB3,EV71,andPVaremembersofthegenusEnterovirus;however,theycausecompletelydifferenttypesof diseases[10,12,26].Inthisstudy,wefoundthatCVB3 andEV71couldefficientlyinduceSGformation.The 2A pro ofbothviruseswereidentifiedasthekeyfortriggeringSGformation.Ourfindingsmayhelpustobetter understandthemechanismbywhichthepicornaviruses initiateSGformation. Accordingtopreviousstudies,TIA1,HuR,G3BP1, andeIF4GwerechosenastheSGmarkersforourobservations.TIA1andHuRhavebeenshowntobeconstantlyrecruitedtoSGsduringPVinfection,while G3BP1andeIF4GcanbedetectedinSGsattheearly stageofthePVinfection,butnotlater[19-21].TofacilitateobservingtheeffectofvirusonSGformation,we Figure3 Aminoacidalignmentof2A pro inPVandCVB3. Identicalresiduesareinwhiteletteringonablackbackground.Theresiduesmarked withasterisks(*)representtheaminoacidsofCVB32A pro thatwouldbemutatedaccordingtoAdditionalfile1:TableS4. Table1TheeIF4Gcleavageactivityobtainedwith differentvariantsofCVB32A pro 2AmutanteIF4Gcleavageactivity* Wild-type+++ D39E+++ L40F+++ S67F++ Y89L+++ Y90L++ V120M+++ G122E+/ D136N+++ *The2Aproteaseactivitywasdeterminedtobeundetectable( ),detectable insomeassays(+/ ),orpositive(+to+++). Wu etal.VirologyJournal 2014, 11 :192 Page5of10 http://www.virologyj.com/content/11/1/192

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firstconstructedaHeLa EGFP-TIA1 celllineconstitutively expressingTIA1.EGFP-TIA1wasdistributedincytoplasmicgranulesinCVB3-andEV71-infectedHeLa EGFP-TIA1 cells(Figures1and4).ThegranuleswerefurtherconfirmedasSGsbecauseHuR,G3BP1,andeIF4Gwerealso foundinthegranules(Figure1).TheSGsemergedas earlyas3hafterthecellswereinfectedwithCVB3or EV71(MOI=10)(Figures1and5),suggestingthatCVB3 andEV71mayefficientlyinduceSGformation.Wealso observedthatG3BP1andeIF4Gwerenotlocalizedtothe SGsatthelatestagesofCVB3orEV71infection(datanot shown),whichwassimilartotheresultsduringPVinfection[19,21].Thus,itislikelythatSGformationmaybe acommonfeatureintheresponsetopicornavirus infections. ItwouldbeinterestingtoknowhowpicornavirusesinitiateSGformation.SGformationmaybetriggereddirectlybyasingleviralcomponent,oritcouldbeindirectly Figure4 Theproteaseactivityisrequiredfor2A pro toinduceSGs.(A) HeLacellswereco-transfectedwithpmCherry-HuRandoneofthe plasmids(pEGFP-2A D39E ,pEGFP-2A L40F ,pEGFP-2A S67F ,pEGFP-2A Y89L ,pEGFP-2A Y90L ,pEGFP-2A V120M ,pEGFP-2A G122E ,orpEGFP-2A D136N ).Thecells wereobservedat24hpost-transfection.ThepercentageofEGFP-expressingcellswithgranuleswasquantifiedasinFigure2A.Theaverageof threeindependentexperimentsisindicatedonthelowerrightofeachpanel. (B) HeLacellsweretransfectedwithpEGFP-C1,orpEGFP-2A G122E . Thecellswerefixedat24hpost-transfectionandthenstainedforG3BP1.NucleiwereidentifiedbyHoechst33342staining.Thelocalizationof themCherry-HuR (A) orG3BP1 (B) proteinswasdeterminedusingafluorescencemicroscope(×400). (C) Theproteaseactivityofthe2A pro mutants basedonthecleavageefficiencyofeIF4Gwasdetermined.HeLacellsweretransfectedwiththese2A pro mutant-expressingplasmidsandtheintegrity ofeIF4Gwasdetectedbywesternblottingat24hpost-transfection. Wu etal.VirologyJournal 2014, 11 :192 Page6of10 http://www.virologyj.com/content/11/1/192

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inducedbythedistortedcellularenvironmentwhenthe picornavirusinfectionsoccur.Uponobservationofthe cellularresponsetoeveryCVB3protein,SGstracedby mCherry-HuRorendogenousHuRcouldonlybeviewed incellsexpressing2A pro (Figure2).HuRdominantlylocalizesinthenucleus.Itisknownthat2A pro candisturbthe nucleus-cytoplasmtraffickingofmacromoleculesthrough cleavingnuclearporeproteins[13].Thus,thetranslocalizationofHuRmaybeaconsequenceofthenucleuscytoplasmtraffickingmalfunctioncausedby2A pro .For thisreason,wevalidatedourobservationusingthecytoplasmicSGmarkerG3BP1.Interestingly,G3BP1-labeled SGscouldalsoonlybeviewedincellsexpressing2A pro (Figure2). Tofurtherverifytheroleof2A pro inSGformation,we generatedeight2A pro mutantsbasedonstudiesofPV 2A pro [20,21,23].Amongthem,theproteaseactivityof 2A G122E wasattenuatedshownbymeasuringthecleavage efficiencyofeIF4G.Accordingly,2A G122E didnotinduce SGformation,indicatingthattheproteaseactivitywasrequiredfor2A pro totriggerSGformation(Figure4).Our resultssuggestthat2A pro maybethekeyviralproteinof CVB3andEV71toinduceSGformation. Theroleof2A pro inSGformationmaybesimilarinPV infection.ArecentstudyshowedthatPVinfectioncould causetheredistributionofSRp20,anucleocytoplasmic shuttlingsplicingfactor[18].Theectopicexpressionof PV2A pro wassufficienttocausethere-localizationof SRp20fromthenucleustothecytoplasmicgranules[18]. AlaterstudydemonstratedthatSRp20couldco-localize withTIA1inthecytoplasmicgranulesofPV-infected cells,suggestingthatSRp20mightalsobeaSGcomponent[22].Theseobservationsprovideextraevidencefor theroleof2A pro inSGformationduringpicornavirus infections. Insummary,ourfindingshelpustobetterunderstand themechanismbywhichpicornavirusesinitiateSGformationininfectedcells.However,thecellularcomponentsthat2A pro targetstotriggerSGformationremain unknown.Furtherstudyisneededtoidentifythesetargetstoelucidatethiscellularevent. Conclusion Inthisstudy,wedemonstratedthatbothCVB3and EV71infectionscaninduceSGformationandthat2A pro playsacrucialroleintheinductionofSGformation duringtheseinfections.Theproteaseactivitywasnecessaryfor2A pro totriggerSGformation.Thisnovelfindingmayhelpustobetterunderstandthemechanismby whichpicornavirusesinitiatetheSGresponse. Methods Celllinesandviruses HeLacellsweregrowninDulbecco ’ smodifiedEagle medium(DMEM)(Invitrogen,Carlsbad,CA)supplementedwith8%(growthmedium)or5%(maintaining Figure5 EV71andits2A pro alsoinduceSGformation.(A) HeLa EGFP-TIA1 cellsweremock-infected,orinfectedwithEV71(MOI=10)for3h. Afterfixation,thecellswerestainedforHuR.ThequantificationofSGformationwasdeterminedbyanalyzingmorethan100cellsthatcontained threeormoregranulesinmultiplefieldsforeachexperiment.Theaverageofthreeindependentexperimentsisindicatedonthelowerrightofeach panel. (B) HeLacellswereco-transfectedwithpmCherry-HuRandpEGFP-EV712AorpEGFP-C1andthecellswerefixedat24hpost-transfectionand thenimaged.NucleiwerestainedwithHoechst33342.ThelocalizationofEGFP-TIA1 (A) ,HuR (A) ,ormCherry-HuR (B) wasdeterminedunder afluorescencemicroscope(×400).Thepercentageofgr eencellspossessingSGswasquantifiedasinFigure2A. Wu etal.VirologyJournal 2014, 11 :192 Page7of10 http://www.virologyj.com/content/11/1/192

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medium)fetalbovineserum(FBS)(BiologicalIndustriesIsrael).AHeLacelllinestablyexpressingEGFPTIA1wasestablishedasdescribedpreviously[27]. HeLacellswereculturedin12-wellplatestoapproximately60%confluenceandtransfectedwiththeplasmid pEGFP-TIA1usingLipofectamine2000(Invitrogen).Cells werethengrowninmediumcontainingG418(500 g/ ml).Wereplacedtheculturemediumevery2days untilcolonieswereobserved.Weacquiredthestable celllineHeLaEGFP-TIA1through8weeksofselection. HeLaEGFP-TIA1cellsstablyexpressingEGFP-TIA1were growninDMEMcontaining10%FBSand500 g/mlG418. TheCVB3WoodruffstrainwaspassagedinHeLacells andtiteredbyplaqueassaysasdescribedpreviously[28]. TheEV71BrCrstrainwaspassagedinVerocellsandtitratedusingthe50%tissuecultureinfectivedose(TCID50) assayasdescribedinourpreviousresearch[29].PlasmidconstructionTheplasmidexpressingEGFP-taggedTIA1oreIF4Gwas constructedinourlaboratory.First,theEGFP-coding sequencewasobtainedfro mpEGFP-N1(Clontech, MountainView,CA)bypolymerasechainreaction(PCR). ThepcDNA3.1(Invitrogen)andEGFPampliconswere digestedby Nhe Iand Hind IIIandthenligatedat16°C overnightwithT4ligase.TheobtainedplasmidwasdesignatedaspEGFP-C1.TIA1andeIF4GcDNAswereamplifiedbyreversetranscriptionPCR(RT-PCR)fromthe RNAextractspreparedfromHeLacellsusingTRIzolreagents(Invitrogen)andthenclonedintopEGFP-C1.The resultingplasmidsweredesignatedaspEGFP-TIA1and pEGFP-eIF4G,respectively.Similarly,thepmCherry-HuR plasmid,encodingafusionproteinofHuRandredfluorescenceproteinmCherry,wasconstructedbasedon pmCherry-C1(Clontech).Theplasmidswereconfirmed byDNAsequencing.Theprimersfortheamplifications arelistedinAdditionalfile1:TableS1. NineplasmidsexpressingEGFP-taggedVP1,VP4VP2-VP3,2Apro,2B,2C,3A,3B,3Cpro,or3DofCVB3 wereconstructedasdescribedpreviously[28,30]and designatedaspEGFP-VP1,pEGFP-VP4-3,pEGFP-2A, pEGFP-2B,pEGFP-2C,pEGFP-3A,pEGFP-3B,pEGFP3C,andpEGFP-3D,respectively.Theprimersarelisted inAdditionalfile1:TableS2andTableS3.Site-directedmutagenesisEightmutantsofCVB32Apro,including2AD39E,2AL40F, 2AS67F,2AY89L,2AY90L,2AV120M,2AG122Eand2AD136N, weregeneratedbyoverlapPCR.Briefly,togenerate 2AD39E,thepEGFP-2ADNAwasamplifiedwith2Asense primerand2AD39Eantisenseprimer,andwith2AD39Esenseprimerand2Aantisenseprimer,respectively (Additionalfile1:TableS3).ThePCRproductswerepurifiedandmixedtogether.Themixturewasamplifiedwith 2Asenseandantisenseprimers.TheresultantDNAwas digestedwith Hind IIIand Xba Iandinsertedintothe cloningsiteofpEGFP-C1.TheseplasmidsweredesignatedaspEGFP-2AD39E,pEGFP-2AL40F,pEGFP-2AS67F, pEGFP-2AY89L,pEGFP-2AY90L,pEGFP-2AV120M,pEGFP2AG122E,andpEGFP-2AD136N,respectively.Thedigested fragmentsweremixedandligatedtopEGFP-C1/ Hind III + Xba I.AllplasmidswereconfirmedbyDNAsequencing.TransfectionForvirusinfection,HeLacellswereseededin24-well platesandgrowntoapproximately60%confluence.The cellswerethenco-transfectedwith0.1 gpmCherryHuRand0.5 gpEGFP-C1,orpEGFP-eIF4Gusing1 l Lipofectamine2000(Invitrogen)perwell.Toexpressthe viralproteins,cellswereseededin24-wellplatesand growntoapproximately70%confluence.Cellswerethen co-transfectedwith0.3 gpmCherry-HuRand0.3 g plasmidencodingEGFP-taggedCVB32Apro,2B,2C,3A, 3B,3Cpro,3D,VP1,VP4-VP2-VP3,2AD39E,2AL40F, 2AS67F,2AY89L,2AY90L,2AV120M,2AG122E,or2AD136N; EV712Apro;ortheemptyvectorpEGFP-C1.Fivehours later,theculturemediawereremovedandreplacedwith freshmedia.ImagesweretakenusinganAxiovert200 fluorescencemicroscope(CarlZeiss,Gottingen,Germany) at24hpost-transfection.Somecellswereseededin48wellplatesandtransfectedwithplasmidencodingEGFPtaggedCVB32Apro,2AG122Eor3Cpro.Controlcellswere transfectedwithpEGFP-C1.After24hpost-transfection, thecellswerefixedforimmunofluorescenceassayofHuR andG3BP1.ToinvestigateeIF4Gcleavage,HeLacells weretransfectedwithplasmidencodingEGFP-tagged CVB32Apro,2AD39E,2AL40F,2AS67F,2AY89L,2AY90L, 2AV120M,2AG122Eor2AD136N.Cellswereharvestedat 24hpost-transfection.Whole-celllysatesweresubjected tosodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)andwesternblottinganalysis.VirusinfectionHeLaEGFP-TIA1cellsweremock-infected,orinfectedwith CVB3orEV71atamultiplicationofinfection(MOI)of 10.At3hp.i.,HeLaEGFP-TIA1cellswerefixedforimmunofluorescenceassays.HeLacells,co-transfected withpmCherry-HuRandpEGFP-C1,orpEGFP-eIF4G orpEGFP-eIF4GG681E,weremock-infectedorinfected withCVB3(MOI=10).At3,4,or6hp.i.,thecellswere washedoncewithphosphate-bufferedsaline(PBS),and processedforimagingusinganAxiovert200fluorescencemicroscope.Arsenite(Ars)treatmentArshasbeenwidelyusedasastimulatorofSGs[2].To induceSGs,cellsweretreatedwithsodiumarseniteWu etal.VirologyJournal 2014, 11 :192 Page8of10 http://www.virologyj.com/content/11/1/192

PAGE 9

(NaArs)(Sigma-Aldrich,St.Louis,MO)ataconcentrationof0.5mMingrowthmediumfor30min.ImmunofluorescenceFollowingvirusinfection,Arstreatment,orplasmidtransfectionasdescribedabove,thecellswerewashedoncewith PBSandthenfixedwith4%paraformaldehydeatroom temperaturefor30min.Thecellsweretreatedwith0.25% TritonX-100for10minandblockedwithPBScontaining 1%bovineserumalbumin(BSA)for30minatroom temperature.Cellswerethenincubatedovernightat4°C withprimaryantibodydilutedinblockingbufferandwith secondaryantibodyfor2hatroomtemperatureinthe dark.G3BP1wasdetectedviaamonoclonalanti-G3BP1 antibody(611126,BDTransductionLabs,SanJose,CA)at adilutionof1:200andaCF555-labeledgoatanti-mouse secondaryantibody(20231,Biotium,Hayward,CA)atadilutionof1:1000.HuRwasdetectedviaapolyclonalantibody(11910-1-AP,ProteintechGroupInc.,Chicago,IL)at adilutionof1:200andaCF555-labeledanti-rabbitsecondaryantibody(20232,Biotium)atadilutionof1:1000.The cellswerethenwashedthreetimeswithPBSandstained withHoechst33342(0.4 g/mlinPBS).Imagesweretaken usinganAxiovert200fluorescencemicroscope.WesternblottingProteinswereextractedfromthetreatedcellsusingPierce RIPABufferwithPMSFcocktail.Approximately2 gof theextractedproteinswereappliedtoSDS-PAGE.The separatedproteinsweretransferredtoapolyvinylidene fluoride(PVDF)membrane(0.45 m,Millipore,Billerica, MA),whichwasblockedwith5%nonfatmilkfor2hat 37°Candincubatedwithprimaryantibodyovernightat 4°C.Apolyclonalrabbitanti-eIF4Gantibody(15704-1-AP, ProteintechGroupInc.)was1:1000dilutedtodetect eIF4G;apolyclonalanti-actinantibody(sc-130301,Santa CruzBiotechnology,SantaCruz,CA)was1:1000dilutedto detect -actin.Afterastandardwashing,themembrane wasincubatedwithhorseradishperoxidase(HRP)-labeled secondaryantibody(ZhongshanGoldenbridgeBiotech, Beijing,China)for1hatroomtemperatureandwashed again.TheblotswerestainedusingSuperSignalkit (Pierce,Rockford,IL)andimagedbyaLAS4000chargecoupledcamera(Fujifilm,Tokyo,Japan).The -actinwas employedasaloadingcontrol.AdditionalfileAdditionalfile1:TableS1. PCRprimersfortheconstructionofplasmids pEGFP-C1,pEGFP-TIA1,pmCherry-HuR,pEGFP-eIF4GandpEGFP-eIF4GG681E. TableS2. PCRprimersfortheconstructionofplasmidsexpressingCVB3 protein2B,3A,3B,3C,and3D. TableS3. PCRprimersequencesusedfor thesite-directedmutagenesisof2Apro. TableS4. TheeIF4Gcleavageactivity obtainedwithdifferentvariantsofPV2Apro[23-25]. FigureS1. Expressionof CVB32AproorEV712AproinVerocellsresultsinSGformation.Verocells wereco-transfectedwithpmCherry-HuRandpEGFP-C1orpEGFP-CVB32A orpEGFP-EV712A.NucleiwereidentifiedbyHoechst33342.The2A expressionandmCherry-HuR-positiveSGsweredeterminedusinga fluorescencemicroscope(×400)at24hpost-transfection. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions SWparticipatedinthedesignofthestudy,performedplasmidconstruction, immunofluorescenceassays,andwesternblottinganddraftedthe manuscript.YWcoordinatedtheexperimentaldesignandhelpededitthe manuscript.LLperformedviralinfectionandcelltransfection.XSparticipated inthedesignofthestudy.TWandXZperformedarsenitetreatment.LT,YL andYCperformedcelltransfection.XLandFZhelpedcoordinatethe experimentaldesign.WZandZZmadesubstantialcontributionstothe experimentaldesignandcoordination.Allauthorsreadandapprovedthe finalmanuscript. Acknowledgments ThisstudywassupportedbytheNaturalScienceFoundationofChina (NSFC)(81271825toZ.Zhong,31270198toW.Zhao,81101234toT.Lei, 81101235toY.Wang,and31300144toT.Wang).WethanktheHeilongjiang ProvincialKeyLaboratoryofPathogensandImmunity,andtheHeilongjiang ProvincialScienceandTechnologyInnovationTeaminHigherEducation InstitutesforInfection,HarbinMedicalUniversity,Harbin150081,Chinafor technicalsupport. Authordetails1DepartmentofMicrobiology,HarbinMedicalUniversity,Harbin150081, China.2DepartmentofCellBiology,HarbinMedicalUniversity,Harbin 150081,China.3DivisionofGastroenterologyandHepatology,Departmentof Medicine,UniversityofFlorida-Jacksonville,Jacksonville,FL32206,USA. Received:12April2014Accepted:26October2014 References1.AndersonP,KedershaN: Stressgranules. CurrBiol 2009, 19: R397 – R398. 2.BuchanJR,ParkerR: Eukaryoticstressgranules:theinsandoutsoftranslation. MolCell 2009, 36: 932 – 941. 3.GilksN,KedershaN,AyodeleM,ShenL,StoecklinG,DemberLM,AndersonP: Stressgranuleassemblyismediatedbyprion-likeaggregationofTIA-1. MolBiolCell 2004, 15: 5383 – 5398. 4.vonRoretzC,DiMarcoS,MazrouiR,GallouziIE: TurnoverofAU-rich-containing mRNAsduringstress:amatterofsurvival. WileyInterdiscipRevRNA 2011, 2: 336 – 347. 5.WhiteJP,LloydRE: Regulationofstressgranulesinvirussystems. TrendsMicrobiol 2012, 20: 175 – 183. 6.LloydRE: Howdovirusesinteractwithstress-associatedRNAgranules? PLoSPathog 2012, 8: e1002741. 7.Valiente-EcheverriaF,MelnychukL,MoulandAJ: Viralmodulationofstress granules. VirusRes 2012, 169: 430 – 437. 8.BorgheseF,MichielsT: Theleaderproteinofcardiovirusesinhibitsstress granuleassembly. JVirol 2011, 85 (18):9614 – 9622. 9.LangereisMA,FengQ,vanKuppeveldFJ: MDA5localizestostress granules,butthislocalizationisnotrequiredfortheinductionoftypeI interferon. JVirol 2013, 87 (11):6314 – 6325. 10.KnowltonKU: CVBinfectionandmechanismsofviralcardiomyopathy. CurrTopMicrobiolImmunol 2008, 323: 315 – 335. 11.FairweatherD,StaffordKA,SungYK: UpdateoncoxsackievirusB3myocarditis. CurrOpinRheumatol 2012, 24: 401 – 407. 12.WongSS,YipCC,LauSK,YuenKY: Humanenterovirus71andhand,foot andmouthdisease. EpidemiolInfect 2010, 138: 1071 – 1089. 13.CastelloA,AlvarezE,CarrascoL: Themultifacetedpoliovirus2Aprotease:regulationofgeneexpression bypicornavirusproteases. JBiomed Biotechnol 2011, 2011: 369648. 14.Kuyumcu-MartinezNM,JoachimsM,LloydRE: Efficientcleavageof ribosome-associatedpoly(A)-bindingproteinbyenterovirus3Cprotease. JVirol 2002, 76: 2062 – 2074.Wu etal.VirologyJournal 2014, 11 :192 Page9of10 http://www.virologyj.com/content/11/1/192

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15.RassmannA,HenkeA,ZobawaM,CarlsohnM,SaluzHP,GrableyS,LottspeichF, MunderT: Proteomealterationsinhumanhostcellsinfectedwith coxsackievirusB3. JGenVirol 2006, 87: 2631 – 2638. 16.ChauDH,YuanJ,ZhangH,CheungP,LimT,LiuZ,SallA,YangD: CoxsackievirusB3proteases2Aand3Cinduceapoptoticcelldeaththrough mitochondrialinjuryandcleavageofeIF4GIbutnotDAP5/p97/NAT1. Apoptosis 2007, 12: 513 – 524. 17.deBreyneS,BonderoffJM,ChumakovKM,LloydRE,HellenCU: Cleavageof eukaryoticinitiationfactoreIF5Bbyenterovirus3Cproteases. Virology 2008, 378: 118 – 122. 18.FitzgeraldKD,ChaseAJ,CathcartAL,TranGP,SemlerBL: Viralproteinase requirementsforthenucleocytoplasmicrelocalizationofcellularsplicing factorSRp20duringpicornavirusinfections. JVirol 2013, 87: 2390 – 2400. 19.WhiteJP,CardenasAM,MarissenWE,LloydRE: Inhibitionofcytoplasmic mRNAstressgranuleformationbyaviralproteinase. CellHostMicrobe 2007, 2: 295 – 305. 20.PiotrowskaJ,HansenSJ,ParkN,JamkaK,SarnowP,GustinKE: Stable formationofcompositionallyuniquestressgranulesinvirus-infected cells. JVirol 2010, 84: 3654 – 3665. 21.WhiteJP,LloydRE: PoliovirusunlinksTIA1aggregationandmRNAstress granuleformation. JVirol 2011, 85: 12442 – 12454. 22.FitzgeraldKD,SemlerBL: Poliovirusinfectioninducestheco-localization ofcellularproteinSRp20withTIA-1,acytoplasmicstressgranuleprotein. VirusRes 2013, 176: 223 – 231. 23.YuSF,LloydRE: Identificationofessentialaminoacidresiduesinthe functionalactivityofpoliovirus2Aprotease. Virology 1991, 182: 615 – 625. 24.BarcoA,VentosoI,CarrascoL: TheyeastSaccharomycescerevisiaeasa geneticsystemforobtainingvariantsofpoliovirusprotease2A. JBiolChem 1997, 272: 12683 – 12691. 25.VentosoI,BarcoA,CarrascoL: Mutationalanalysisofpoliovirus2Apro. Distinctinhibitoryfunctionsof2Aproontranslationandtranscription. JBiolChem 1998, 273: 27960 – 27967. 26.MuellerS,WimmerE,CelloJ: Poliovirusandpoliomyelitis:ataleofguts, brains,andanaccidentalevent. VirusRes 2005, 111: 175 – 193. 27.WuS,LinL,ZhaoW,LiX,WangY,SiX,WangT,WuH,ZhaiX,ZhongX, GaoS,TongL,XuZ,ZhongZ: AUF1isrecruitedtothestressgranules inducedbycoxsackievirusB3. VirusRes 2014, 192: 52 – 61. 28.WangL,QinY,TongL,WuS,WangQ,JiaoQ,GuoZ,LinL,WangR,ZhaoW, ZhongZ: MiR-342-5psuppressescoxsackievirusB3biosynthesisby targetingthe2C-codingregion. AntiviralRes 2012, 93: 270 – 279. 29.GuoZ,ZhongX,LinL,WuS,WangT,ChenY,ZhaiX,WangY,WuH,TongL, HanY,PanB,PengY,SiX,ZhangF,ZhaoW,ZhongZ: A3Cpro-dependent bioluminescenceimagingassayforinvivoevaluationofanti-enterovirus 71agents. AntiviralRes 2014, 101: 82 – 92. 30.WangT,YuB,LinL,ZhaiX,HanY,QinY,GuoZ,WuS,ZhongX,WangY, TongL,ZhangF,SiX,ZhaoW,ZhongZ: Afunctionalnuclearlocalization sequenceintheVP1capsidproteinofcoxsackievirusB3. Virology 2012, 433: 513 – 521.doi:10.1186/s12985-014-0192-1 Citethisarticleas: Wu etal. : Protease2Ainducesstressgranule formationduringcoxsackievirusB3andenterovirus71infections. VirologyJournal 2014 11 :192. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Wu etal.VirologyJournal 2014, 11 :192 Page10of10 http://www.virologyj.com/content/11/1/192


Wu
et
al.
Virology
Journal
2014,
11:192
http://www.virologyj.com/content/11/1/192



RESEARCHOpenAccess
Protease
2A
induces
stress
granule
formation
during
coxsackievirus
B3
and
enterovirus
71
infections


Shuo
Wu1,
Yan
Wang1,
Lexun
Lin1,
Xiaoning
Si1,
Tianying
Wang1,
Xiaoyan
Zhong1,
Lei
Tong1,
Ying
Luan1,
Yang
Chen1,
Xiaoyu
Li3,
Fengmin
Zhang1,
Wenran
Zhao2*
and
Zhaohua
Zhong1*



Abstract


Background:
Stress
granules
(SGs)
are
granular
aggregates
in
the
cytoplasm
that
are
formed
under
a
variety
of
stress
situations
including
viral
infection.
Previous
studies
indicate
that
poliovirus,
a
member
of
Picornaviridae,
can
induce
SG
formation.
However,
the
exact
mechanism
by
which
the
picornaviruses
induce
SG
formation
is
unknown.


Method:
The
localization
of
SG
markers
in
cells
infected
with
coxsackievirus
B3
(CVB3)
or
enterovirus
71
(EV71)
and
in
cells
expressing
each
viral
protein
was
determined
via
immunofluorescence
assays
or
plasmid
transfection.
Eight
plasmids
expressing
mutants
of
the
2A
protease
(2Apro)
of
CVB3
were
generated
using
a
site-directed
mutagenesis
strategy.
The
cleavage
efficiencies
of
eIF4G
by
CVB3
2Apro
and
its
mutants
were
determined
via
western
blotting
assays.


Results:
In
this
study,
we
found
that
CVB3
infection
induced
SG
formation,
as
evidenced
by
the
co-localization
of
some
accepted
SG
markers
in
viral
infection-induced
granules.
Furthermore,
we
identified
that
2Apro
of
CVB3
was
the
key
viral
component
that
triggered
SG
formation.
A
2Apro
mutant
with
the
G122E
mutation,
which
exhibited
very
low
cleavage
efficiency
toward
eIF4G,
significantly
attenuated
its
capacity
for
SG
induction,
indicating
that
the
protease
activity
was
required
for
2Apro
to
initiate
SG
formation.
Finally,
we
observed
that
SGs
also
formed
in
EV71-infected
cells.
Expression
of
EV71
2Apro
alone
was
also
sufficient
to
cause
SG
formation.


Conclusion:
Both
CVB3
and
EV71
infections
can
induce
SG
formation,
and
2Apro
plays
a
crucial
role
in
the
induction
of
SG
formation
during
these
infections.
This
finding
may
help
us
to
better
understand
how
picornaviruses
initiate
the
SG
response.


Keywords:
Stress
granule,
2A
protease,
Coxsackievirus
B,
Enterovirus
71



Background


Stress
granules
(SGs)
are
granular
aggregates
formed
in
the
cytoplasm
of
eukaryotic
cells
exposed
to
a
variety
of
environmental
stress
conditions,
e.g.,
heat
shock,
UV
irradiation,
hypoxia,
endoplasmic
reticulum
stress,
and
viral
infection
[1,2].
SGs
typically
contain
translationally
silent
mRNAs,
40S
ribosomal
subunits,
eukaryotic
initiation
factors
(eIFs)
such
as
eIF4E,
eIF4G,
eIF4A,
eIF4B,
eIF3,
and
eIF2,
and
RNA-binding
proteins
(RBPs),
including


*
Correspondence:
zhaowr@ems.hrbmu.edu.cn;
zhongzh@hrbmu.edu.cn
2Department
of
Cell
Biology,
Harbin
Medical
University,
Harbin
150081,
China
1Department
of
Microbiology,
Harbin
Medical
University,
Harbin
150081,
China
Full
list
of
author
information
is
available
at
the
end
of
the
article
poly(A)-binding
protein
(PABP1),
the
embryonic
lethal
abnormal
vision
(ELAV)
Hu
protein
(HuR),
polysomal
ribonuclease
1
(PMR-1),
tristetraprolin
(TTP),
T-cellrestricted
intracellular
antigen
1
(TIA1),
TIA-1-related
protein
(TIAR),
fragile
X
mental
retardation
protein
(FMRP),
and
Ras-Gap
SH3-binding
protein
(G3BP1)
[1,2].
Among
these
recruited
proteins,
HuR,
TIA1,
and
G3BP1
can
act
as
markers
of
SGs
[1-4].


Numerous
viruses
have
been
shown
to
interact
with
SGs
with
different
effects
[5-7].
Some
viruses
induce
stable
SG
formation
during
infection,
e.g.,
respiratory
syncytial
virus
(RSV).
Some
viruses,
e.g.,
mammalian
orthoreoviruses
(MRV),
Semliki
Forest
viruses
(SFV),
and
hepatitis
C
viruses
(HCV)
have
been
shown
to
induce
SG



�
2014
Wu
et
al.;
licensee
BioMed
Central
Ltd.
This
is
an
Open
Access
article
distributed
under
the
terms
of
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Creative
Commons
Attribution
License
(http://creativecommons.org/licenses/by/4.0),
which
permits
unrestricted
use,
distribution,
and
reproduction
in
any
medium,
provided
the
original
work
is
properly
credited.
The
Creative
Commons
Public
Domain
Dedication
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(http://creativecommons.org/publicdomain/zero/1.0/)
applies
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in
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article,
unless
otherwise
stated.



Wu
et
al.
Virology
Journal
2014,
11:192
Page
2
of
10
http://www.virologyj.com/content/11/1/192


formation
but
to
disassemble
these
granules
as
infection
proceeds.
Poliovirus
(PV)
induces
SG
formation
early
during
infection
and
later
inhibits
SGs
through
the
viral
3Cpro-mediated
cleavage
of
G3BP1.
Meanwhile,
TIA1containing
foci
devoid
of
other
SG-defining
components,
such
as
initiation
factors
and
most
mRNAs
were
observed
late
post-infection.
Some
viruses,
e.g.,
West
Nile
virus
(WNV),
dengue
virus
(DV),
rhesus
rotavirus
(RRV),
type
1
human
T-cell
leukemia
viruses
(HTLV-1),
and
human
immunodeficiency
viruses
(HIV)
were
found
to
be
able
to
suppress
SG
formation
[5-7].
Cardioviruses
with
mutant
leader
(L)
proteins,
such
as
Theiler�s
murine
encephalomyelitis
virus
(TMEV),
encephalomyocarditis
virus
(EMCV)
and
Saffold
virus
(SAFV)
induce
SG
formation
throughout
the
infection,
while
expression
of
the
L
protein
during
infection
efficiently
blocks
SG
formation.
Similarly,
influenza
A
virus
(IAV)
infection
fails
to
induce
SGs
unless
viruses
with
NS1
mutations
are
used
[5-9].
In
some
cases,
SGs
play
an
important
role
in
host
antiviral
defense,
e.g.,
IAV,
MRV
and
vaccinia
virus
(VV).
However,
SGs
induced
by
RSV
can
promote
virus
replication
[5-7].


Group
B
coxsackieviruses
(CVBs)
are
the
major
pathogens
of
human
viral
myocarditis
and
dilated
cardiomyopathy
[10,11].
Enterovirus
71
(EV71)
is
the
major
pathogen
of
human
hand,
foot,
and
mouth
disease
(HFMD)
[12].
Both
CVBs
and
EV71
belong
to
the
enterovirus
genus
of
the
Picornaviridae
family.
The
picornavirus
genome
is
a
~7.0-kb-to-8.5-kb
single-stranded
positive-sense
RNA
(+ssRNA)
that
is
composed
of
a
single
open
reading
frame
(ORF)
and
two
untranslated
regions
(UTRs)
at
its
5'
and
3'
flanks.
The
genome
can
act
as
an
mRNA
encoding
a
polyprotein
that
is
proteolytically
processed
by
viral
proteinases
into
structural
and
nonstructural
proteins
[13].
Picornaviruses
induce
multiple
alterations
in
host
cells
to
facilitate
its
replication.
Suppressing
cellular
biosynthesis
by
viral
proteinases
2A
(2Apro)
and
3C
(3Cpro)isone
of
the
most
notable
alterations
[13-17].
Upon
the
cleavage
of
eIF4G,
eIF5B,
and
PABP
by
3Cpro
and
2Apro,picornaviruses
can
shut
off
cap-dependent
translation
and
terminate
cellular
biosynthesis
[13-17].
In
addition,
through
the
cleavage
of
nuclear
pore
complex
proteins
(Nups),
2Apro
can
alter
RNA
and
protein
trafficking
between
the
nucleus
and
cytoplasm
[13].
A
recent
report
shows
that
2Apro
of
PV,
a
member
of
Picornaviridae,
can
cause
a
dramatic
nuclear-cytoplasm
re-localization
of
SRp20,
a
cellular
splicing
factor
that
is
also
defined
as
an
IRES
trans-acting
factor
(ITAF)
[18].


Recent
studies
indicate
that
PV
induces
unique
SGs
in
infected
cells
[19-21].
The
PV-induced
SGs
contain
certain
components
that
do
not
localize
to
SGs
induced
by
oxidative
stress,
e.g.,
Sam68
and
SRp20
[20,22].
However,
the
exact
mechanism
by
which
the
picornavirus
induces
SG
formation
is
unknown.
In
this
study,
we
confirmed
that
CVB
type
3
(CVB3)
and
EV71
also
induce
SG


formation.
We
found
that
2Apro
plays
a
crucial
role
in
SG
induction
because
2Apro
alone
is
sufficient
to
trigger
SG
formation.
Our
findings
may
help
us
to
better
understand
the
mechanism
by
which
picornaviruses
initiate
SG
formation.


Results


CVB3
infection
induces
SG
formation


To
facilitate
observing
SG
formation
during
CVB3
infection,
we
constructed
a
HeLa
cell
line
(HeLaEGFP-TIA1),
constitutively
expressing
EGFP-TIA1.
SG
formation
was
first
determined
by
observing
the
expression
and
localization
of
TIA1
and
HuR.
HeLaEGFP-TIA1
cells
were
infected
with
CVB3
(MOI
=
10)
or
treated
with
0.5
mM
NaArs
for
30
min.
In
the
mock-treated
cells,
EGFP-TIA1
was
distributed
in
both
the
cytoplasm
and
nucleus.
In
the
Ars-treated
and
CVB3-infected
cells,
EGFP-TIA1
was
granularly
distributed
in
the
cytoplasm.
The
granules
began
to
emerge
in
the
cytoplasm
of
the
CVB3-infected
cells
at
3
h
post-infection
(p.i.)
(Figure
1).


Using
an
anti-HuR
or
anti-G3BP1
antibody,
we
found
that
HuR
was
predominantly
localized
in
the
nucleus
of
the
mock-infected
cells,
but
it
re-localized
to
the
cytoplasmic
granules
after
both
Ars
treatment
and
CVB3
infection
(Figure
1A).
G3BP1
was
distributed
evenly
in
the
cytoplasm
of
the
mock-infected
cells
but
formed
granules
in
the
CVB3-infected
cells
at
3
h
p.i.
but
the
G3BP1-positive
granules
dispersed
at
6
h
p.i.
(Figure
1B).
EGFP-TIA1
and
HuR
were
co-localized
in
the
granules
in
Ars-treated
and
CVB3-infected
cells
(Figure
1A).
G3BP1
was
also
colocalized
with
EGFP-TIA1
in
the
granules
of
Ars-treated
(data
not
shown)
and
CVB3-infected
cells
(Figure
1B).


Furthermore,
in
HeLa
cells
co-transfected
with
pEGFP-eIF4G
and
pmCherry-HuR,
both
mCherry-HuR
and
EGFP-eIF4G
re-located
from
the
nucleus
to
the
cytoplasmic
granules
after
CVB3
infection
(MOI
=
10)
for
3
h
(Figure
1C).
We
also
noticed
that
the
SGs
contained
HuR
but
no
eIF4G
at
6
h
p.i.
(Figure
1C).
These
data
demonstrate
that
CVB3
induced
typical
SGs.


2Apro
is
the
key
protein
that
triggers
SG
formation


To
identify
the
viral
component
that
triggered
SG
formation
during
CVB3
infection,
HeLa
cells
were
cotransfected
with
pmCherry-HuR
and
one
of
the
nine
plasmids
expressing
EGFP-tagged
viral
proteins
(VP1,
VP4-3,
2Apro,2B,
2C,3A,
3B,3Cpro,
and
3D).
We
observed
that
mCherry-HuR
was
translocated
from
the
nucleus
to
the
cytoplasmic
granules
in
cells
transfected
with
pEGFP-2A
after
24
h
post-transfection
(Figure
2A).
We
observed
a
similar
result
in
Vero
cells
(Additional
file
1:
Figure
S1).
The
nuclear
localization
of
mCherry-HuR
was
not
affected
in
cells
expressing
EGFP,
VP1,
VP4-3,
2B,
2C,
3A,
3B,
3Cpro,
or
3D
(Figure
2A).
Quantification
of
these
experiments
indicated
that
93%
(�2%
[standard
deviation])



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Figure
1
CVB3
infection
induces
SG
formation.
(A)
HeLaEGFP-TIA1
cells
were
mock
infected,
exposed
to
0.5
mM
NaArs
for
30
min,
or
infected
with
CVB3
(MOI
=10)
for
3
h.
The
cells
were
then
fixed
and
stained
for
HuR.
(B)
HeLaEGFP-TIA1
cells
were
infected
with
CVB3
as
discribed
in
(A).The
cells
were
then
fixedand
stainedfor
G3BP1.
(C)
HeLa
cells,
co-transfected
with
pEGFP-eIF4G
and
pmCherry-HuR,
were
infected
with
CVB3
as
discribed
in
(A).
The
cells
were
then
fixed
and
imaged.
Nuclei
were
identified
by
Hoechst
33342
staining.
Cells
were
examined
using
a
fluorescence
microscopy
(�400).



of
CVB3
2Apro-expressing
cells
exhibited
SGs,
while
By
monitoring
the
endogenous
HuR
distribution
using
the
percentages
of
SGs
in
other
groups
of
cells
did
not
the
anti-HuR
antibody,
a
similar
result
was
observed
in
exceed
5%.
cells
only
transfected
with
the
viral
protein-expressing



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Figure
2
CVB3
2Apro
is
identified
as
the
viral
component
that
induces
SG
formation
in
HeLa
cells.
(A)
HeLa
cells
were
co-transfected
with
the
pmCherry-HuR
plasmid
and
one
of
the
following
plasmids:
pEGFP-2A,
pEGFP-2B,
pEGFP-2C,
pEGFP-3A,
pEGFP-3B,
pEGFP-3C,
pEGFP-3D,
pEGFP-VP1,
or
pEGFP-VP4-3.
At
24
h
post-transfection,
the
cells
were
imaged.
The
quantification
of
SG
formation
for
each
group
was
determined
by
analyzing
the
percentage
of
EGFP-expressing
cells
that
contained
three
or
more
granules.
At
least
100
cells
were
counted
for
each
experiment.
The
average
of
three
independent
experiments
is
indicated
on
the
lower
part
of
each
panel.
(B)
HeLa
cells
were
transfected
with
pEGFP-C1,
pEGFP-2A,
or
pEGFP-3C.
The
cells
were
then
fixed
at
24
h
post-transfection
and
stained
for
HuR
via
an
anti-HuR
rabbit
polyclonal
antibody
and
a
CF555-labeled
goat
anti-rabbit
secondary
antibody.
(C)
HeLa
cells
were
treated
as
discribed
in
(B)
and
then
stained
for
G3BP1
via
an
anti-G3BP1
mouse
monoclonal
antibody
and
a
CF555-labeled
goat
anti-mouse
secondary
antibody.
Hoechst
33342
was
used
to
counterstain
the
nuclei.
The
SGs
were
determined
by
observing
the
granular
distribution
of
mCherry-HuR
(A),
HuR
(B),
and
G3BP1
(C)
in
the
cytoplasm
under
a
fluorescence
microscope
(�400).
The
cytoplasmic
granules
are
indicated
by
arrow
heads.



plasmids
(Figure
2B).
Identically,
G3BP1
detection
showed
that
G3BP1
was
distributed
in
cytoplasmic
granules
only
in
the
cells
expressing
2Apro
(Figure
2C).
These
results
imply
that
2Apro
is
the
key
viral
component
that
triggers
SG
formation
during
CVB3
infection.


The
protease
activity
is
necessary
for
2Apro
to
trigger
SG
formation


Because
the
amino
acid
sequences
of
CVB3
2Apro
and
PV
2Apro


were
highly
homologous
(Figure
3),
eight
EGFP-
tagged
CVB3
2Apro
mutants
including
D39E,
L40F,
S67F,


Y89L,
Y90L,
V120M,
G122E,
and
D136N
(marked
with
asterisks
in
Figure
3)
were
generated
according
to
the
PV
2Apro
variants
that
exhibited
different
cleavage
activity
toward
eIF4G
(Additional
file
1:
Table
S4).
Their
protease
activities,
as
indicated
by
their
efficiency
in
cleaving
eIF4G
[23-25],
were
evaluated.
Table
1
shows
the
results
of
their
assessment
of
eIF4G
cleavage
activity.
In
cells
expressing
these
mutants,
only
2AG122E
showed
attenuated
protease
activity
compared
with
the
prototype
(Figure
4C
and
Table
1).
The
mutants
including
L40F,
S67F,
V120M,
G122E,
and
D136N
behaved
as
expected
[24,25],
while
the



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Figure
3
Amino
acid
alignment
of
2Apro
in
PV
and
CVB3.
Identical
residues
are
in
white
lettering
on
a
black
background.
The
residues
marked
with
asterisks
(*)
represent
the
amino
acids
of
CVB3
2Apro
that
would
be
mutated
according
to
Additional
file
1:
Table
S4.



mutants
D39E,
Y89L,
and
Y90L
did
not
appear
to
behave
as
expected
[23].


To
determine
whether
the
proteinase
activity
of
2Apro
is
required
for
SG
formation,
HeLa
cells
were
co-transfected
with
pmCherry-HuR
and
each
of
the
mutant-expressing
plasmids.
We
found
that
only
a
few
SGs
were
formed
in
cells
expressing
2AG122E.
In
contrast,
there
were
abundant
SGs
formed
in
the
cells
expressing
other
2Apro
mutants
(Figure
4A).
Quantitative
analysis
of
these
data
revealed
that
only
7%
(�3%)
of
EGFP-2AG122E-expressing
cells
contained
SGs
and
over
90%
of
cells
expressing
the
other
2A
mutants
remained
positive
for
SGs.
Similar
results
were
observed
for
G3BP1
as
the
SG
marker
(Figure
4B).
Therefore,
we
concluded
that
the
protease
activity
was
required
for
2Apro
to
induce
SGs.


EV71
and
its
2Apro
also
induce
SG
formation


We
next
examined
whether
SGs
formed
in
cells
infected
with
EV71.
HeLaEGFP-TIA1
cells
were
mock-infected
or
infected
with
EV71
at
an
MOI
of
10.
In
the
infected
cells,


Table
1
The
eIF4G
cleavage
activity
obtained
with
different
variants
of
CVB3
2Apro


2A
mutant
eIF4G
cleavage
activity*
Wild-type
+
+
+
D39E
+
+
+
L40F
+
+
+
S67F
+
+
Y89L
+
+
+
Y90L
+
+
V120M
+
+
+
G122E
+/-
D136N
+
+
+


*The
2A
protease
activity
was
determined
to
be
undetectable
(-),
detectable
in
some
assays
(+/-),
or
positive
(+
to
+
+
+).


both
EGFP-TIA1
and
HuR
underwent
redistribution
and
co-localized
to
the
cytoplasmic
granules
(Figure
5A).
At
3
h
p.i.,
approximately
34%
of
the
infected
cells
showed
granules.
The
granules
also
contained
G3BP1
and
eIF4G
(data
not
shown),
indicating
that
EV71
infection
also
induced
SG
formation.
Similar
to
the
experiments
described
in
the
previous
section,
HeLa
cells
were
also
co-transfected
with
pmCherry-HuR
and
pEGFP-C1
or
pEGFP-EV71
2A.
We
found
that
91%
(�4%)
of
EV71
2Apro-expressed
cells
were
positive
for
SGs
(Figure
5B).
A
similar
result
was
obtained
in
the
infected
Vero
cells
(Additional
file
1:
Figure
S1).
Thus,
EV71
2Apro
can
function
in
SG
formation.


Discussion


The
interaction
between
viruses
and
host
cells
is
critical
for
understanding
the
pathogenesis
of
viral
infections.
SG
formation
is
one
of
the
remarkable
cellular
events
when
cells
sense
an
unfavorable
stimulation
[1,2].
Multiple
viruses
interact
with
SGs
in
different
ways
[5-7].
It
has
been
reported
that
PV
can
induce
SG
formation.
CVB3,
EV71,
and
PV
are
members
of
the
genus
Enterovirus;
however,
they
cause
completely
different
types
of
diseases
[10,12,26].
In
this
study,
we
found
that
CVB3
and
EV71
could
efficiently
induce
SG
formation.
The
2Apro
of
both
viruses
were
identified
as
the
key
for
triggering
SG
formation.
Our
findings
may
help
us
to
better
understand
the
mechanism
by
which
the
picornaviruses
initiate
SG
formation.


According
to
previous
studies,
TIA1,
HuR,
G3BP1,
and
eIF4G
were
chosen
as
the
SG
markers
for
our
observations.
TIA1
and
HuR
have
been
shown
to
be
constantly
recruited
to
SGs
during
PV
infection,
while
G3BP1
and
eIF4G
can
be
detected
in
SGs
at
the
early
stage
of
the
PV
infection,
but
not
later
[19-21].
To
facilitate
observing
the
effect
of
virus
on
SG
formation,
we



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Figure
4
The
protease
activity
is
required
for
2Apro
to
induce
SGs.
(A)
HeLa
cells
were
co-transfected
with
pmCherry-HuR
and
one
of
the
plasmids
(pEGFP-2AD39E,
pEGFP-2AL40F,
pEGFP-2AS67F,
pEGFP-2AY89L,
pEGFP-2AY90L,
pEGFP-2AV120M,
pEGFP-2AG122E,
or
pEGFP-2AD136N).
The
cells
were
observed
at
24
h
post-transfection.
The
percentage
of
EGFP-expressing
cells
with
granules
was
quantified
as
in
Figure
2A.
The
average
of
three
independent
experiments
is
indicated
on
the
lower
right
of
each
panel.
(B)
HeLa
cells
were
transfected
with
pEGFP-C1,
or
pEGFP-2AG122E.
The
cells
were
fixed
at
24
h
post-transfection
and
then
stained
for
G3BP1.
Nuclei
were
identified
by
Hoechst
33342
staining.
The
localization
of
the
mCherry-HuR
(A)
or
G3BP1
(B)
proteins
was
determined
using
a
fluorescence
microscope
(�400).
(C)
The
protease
activity
of
the
2Apro
mutants
based
on
the
cleavage
efficiency
of
eIF4G
was
determined.
HeLa
cells
were
transfected
with
these
2Apro
mutant-expressing
plasmids
and
the
integrity
of
eIF4G
was
detected
by
western
blotting
at
24
h
post-transfection.



first
constructed
a
HeLaEGFP-TIA1
cell
line
constitutively
expressing
TIA1.
EGFP-TIA1
was
distributed
in
cytoplasmic
granules
in
CVB3-and
EV71-infected
HeLaEGFP-TIA1
cells
(Figures
1
and
4).
The
granules
were
further
confirmed
as
SGs
because
HuR,
G3BP1,
and
eIF4G
were
also
found
in
the
granules
(Figure
1).
The
SGs
emerged
as
early
as
3
h
after
the
cells
were
infected
with
CVB3
or
EV71
(MOI
=
10)
(Figures
1
and
5),
suggesting
that
CVB3
and
EV71
may
efficiently
induce
SG
formation.
We
also


observed
that
G3BP1
and
eIF4G
were
not
localized
to
the
SGs
at
the
late
stages
of
CVB3
or
EV71
infection
(data
not
shown),
which
was
similar
to
the
results
during
PV
infection
[19,21].
Thus,
it
is
likely
that
SG
formation
may
be
a
common
feature
in
the
response
to
picornavirus
infections.


It
would
be
interesting
to
know
how
picornaviruses
initiate
SG
formation.
SG
formation
may
be
triggered
directly
by
a
single
viral
component,
or
it
could
be
indirectly



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Figure
5
EV71
and
its
2Apro
also
induce
SG
formation.
(A)
HeLaEGFP-TIA1
cells
were
mock-infected,
or
infected
with
EV71
(MOI
=10)
for
3
h.
After
fixation,
the
cells
were
stained
for
HuR.
The
quantification
of
SG
formation
was
determined
by
analyzing
more
than
100
cells
that
contained
three
or
more
granules
in
multiple
fields
for
each
experiment.
The
average
of
three
independent
experiments
is
indicated
on
the
lower
right
of
each
panel.
(B)
HeLa
cells
were
co-transfected
with
pmCherry-HuR
and
pEGFP-EV71
2A
or
pEGFP-C1
and
the
cells
were
fixed
at
24
h
post-transfection
and
then
imaged.
Nuclei
were
stained
with
Hoechst
33342.
The
localization
of
EGFP-TIA1
(A),HuR
(A),ormCherry-HuR
(B)
was
determined
under
a
fluorescence
microscope
(�400).
The
percentage
of
green
cells
possessing
SGs
was
quantified
as
in
Figure
2A.



induced
by
the
distorted
cellular
environment
when
the
picornavirus
infections
occur.
Upon
observation
of
the
cellular
response
to
every
CVB3
protein,
SGs
traced
by
mCherry-HuR
or
endogenous
HuR
could
only
be
viewed
in
cells
expressing
2Apro
(Figure
2).
HuR
dominantly
localizes
in
the
nucleus.
It
is
known
that
2Apro
can
disturb
the
nucleus-cytoplasm
trafficking
of
macromolecules
through
cleaving
nuclear
pore
proteins
[13].
Thus,
the
translocalization
of
HuR
may
be
a
consequence
of
the
nucleus-
cytoplasm
trafficking
malfunction
caused
by
2Apro.For
this
reason,
we
validated
our
observation
using
the
cytoplasmic
SG
marker
G3BP1.
Interestingly,
G3BP1-labeled
SGs
could
also
only
be
viewed
in
cells
expressing
2Apro
(Figure
2).


To
further
verify
the
role
of
2Apro
in
SG
formation,
we
generated
eight
2Apro
mutants
based
on
studies
of
PV
2Apro


[20,21,23].
Among
them,
the
protease
activity
of
2AG122E
was
attenuated
shown
by
measuring
the
cleavage
efficiency
of
eIF4G.
Accordingly,
2AG122E
did
not
induce
SG
formation,
indicating
that
the
protease
activity
was
required
for
2Apro
to
trigger
SG
formation
(Figure
4).
Our
results
suggest
that
2Apro
may
be
the
key
viral
protein
of
CVB3
and
EV71
to
induce
SG
formation.


The
role
of
2Apro
in
SG
formation
may
be
similar
in
PV
infection.
A
recent
study
showed
that
PV
infection
could
cause
the
redistribution
of
SRp20,
a
nucleocytoplasmic
shuttling
splicing
factor
[18].
The
ectopic
expression
of
PV
2Apro
was
sufficient
to
cause
the
re-localization
of


SRp20
from
the
nucleus
to
the
cytoplasmic
granules
[18].
A
later
study
demonstrated
that
SRp20
could
co-localize
with
TIA1
in
the
cytoplasmic
granules
of
PV-infected
cells,
suggesting
that
SRp20
might
also
be
a
SG
component
[22].
These
observations
provide
extra
evidence
for
the
role
of
2Apro
in
SG
formation
during
picornavirus
infections.


In
summary,
our
findings
help
us
to
better
understand
the
mechanism
by
which
picornaviruses
initiate
SG
formation
in
infected
cells.
However,
the
cellular
components
that
2Apro
targets
to
trigger
SG
formation
remain
unknown.
Further
study
is
needed
to
identify
these
targets
to
elucidate
this
cellular
event.


Conclusion


In
this
study,
we
demonstrated
that
both
CVB3
and
EV71
infections
can
induce
SG
formation
and
that
2Apro
plays
a
crucial
role
in
the
induction
of
SG
formation
during
these
infections.
The
protease
activity
was
necessary
for
2Apro
to
trigger
SG
formation.
This
novel
finding
may
help
us
to
better
understand
the
mechanism
by
which
picornaviruses
initiate
the
SG
response.


Methods


Cell
lines
and
viruses


HeLa
cells
were
grown
in
Dulbecco�s
modified
Eagle
medium
(DMEM)
(Invitrogen,
Carlsbad,
CA)
supplemented
with
8%
(growth
medium)
or
5%
(maintaining



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medium)
fetal
bovine
serum
(FBS)
(Biological
Industries
Israel).
A
HeLa
cell
line
stably
expressing
EGFPTIA1
was
established
as
described
previously
[27].
HeLa
cells
were
cultured
in
12-well
plates
to
approximately
60%
confluence
and
transfected
with
the
plasmid
pEGFP-TIA1
using
Lipofectamine
2000
(Invitrogen).
Cells
were
then
grown
in
medium
containing
G418
(500
�g/
ml).
We
replaced
the
culture
medium
every
2
days
until
colonies
were
observed.
We
acquired
the
stable
cell
line
HeLaEGFP-TIA1
through
8
weeks
of
selection.


HeLaEGFP-TIA1


cells
stably
expressing
EGFP-TIA1
were
grown
in
DMEM
containing
10%
FBS
and
500
�g/ml
G418.
The
CVB3
Woodruff
strain
was
passaged
in
HeLa
cells
and
titered
by
plaque
assays
as
described
previously
[28].
The
EV71
BrCr
strain
was
passaged
in
Vero
cells
and
titrated
using
the
50%
tissue
culture
infective
dose
(TCID50)
assay
as
described
in
our
previous
research
[29].


Plasmid
construction


The
plasmid
expressing
EGFP-tagged
TIA1
or
eIF4G
was
constructed
in
our
laboratory.
First,
the
EGFP-coding
sequence
was
obtained
from
pEGFP-N1
(Clontech,
Mountain
View,
CA)
by
polymerase
chain
reaction
(PCR).
The
pcDNA3.1
(Invitrogen)
and
EGFP
amplicons
were
digested
by
Nhe
Iand
Hind
III
and
then
ligated
at
16�C
overnight
with
T4
ligase.
The
obtained
plasmid
was
designated
as
pEGFP-C1.
TIA1
and
eIF4G
cDNAs
were
amplified
by
reverse
transcription
PCR
(RT-PCR)
from
the
RNA
extracts
prepared
from
HeLa
cells
using
TRIzol
reagents
(Invitrogen)
and
then
cloned
into
pEGFP-C1.
The
resulting
plasmids
were
designated
as
pEGFP-TIA1
and
pEGFP-eIF4G,
respectively.
Similarly,
the
pmCherry-HuR
plasmid,
encoding
a
fusion
protein
of
HuR
and
red
fluorescence
protein
mCherry,
was
constructed
based
on
pmCherry-C1
(Clontech).
The
plasmids
were
confirmed
by
DNA
sequencing.
The
primers
for
the
amplifications
are
listed
in
Additional
file
1:
Table
S1.


Nine
plasmids
expressing
EGFP-tagged
VP1,
VP4VP2-
VP3,
2Apro,
2B,
2C,
3A,
3B,
3Cpro,
or
3D
of
CVB3
were
constructed
as
described
previously
[28,30]
and
designated
as
pEGFP-VP1,
pEGFP-VP4-3,
pEGFP-2A,
pEGFP-2B,
pEGFP-2C,
pEGFP-3A,
pEGFP-3B,
pEGFP3C,
and
pEGFP-3D,
respectively.
The
primers
are
listed
in
Additional
file
1:
Table
S2
and
Table
S3.


Site-directed
mutagenesis


Eight
mutants
of
CVB3
2Apro,
including
2AD39E,2AL40F
,


2AS67F,2AY89L,2AY90L,2AV120M,2AG122E


and
2AD136N
,
were
generated
by
overlap
PCR.
Briefly,
to
generate
2AD39E,
the
pEGFP-2A
DNA
was
amplified
with
2A
sense
primer
and
2AD39E
antisense
primer,
and
with
2AD39E
sense
primer
and
2A
antisense
primer,
respectively
(Additional
file
1:
Table
S3).
The
PCR
products
were
purified
and
mixed
together.
The
mixture
was
amplified
with


2A
sense
and
antisense
primers.
The
resultant
DNA
was
digested
with
Hind
III
and
Xba
I
and
inserted
into
the
cloning
site
of
pEGFP-C1.
These
plasmids
were
designated
as
pEGFP-2AD39E,pEGFP-2AL40F
,
pEGFP-2AS67F
,
pEGFP-2AY89L,
pEGFP-2AY90L,pEGFP-2AV120M,pEGFP2AG122E,
and
pEGFP-2AD136N,
respectively.
The
digested
fragments
were
mixed
and
ligated
to
pEGFP-C1/Hind
III
+
Xba
I.
All
plasmids
were
confirmed
by
DNA
sequencing.


Transfection


For
virus
infection,
HeLa
cells
were
seeded
in
24-well
plates
and
grown
to
approximately
60%
confluence.
The
cells
were
then
co-transfected
with
0.1
�g
pmCherry-
HuR
and
0.5
�g
pEGFP-C1,
or
pEGFP-eIF4G
using
1
�l
Lipofectamine
2000
(Invitrogen)
per
well.
To
express
the
viral
proteins,
cells
were
seeded
in
24-well
plates
and
grown
to
approximately
70%
confluence.
Cells
were
then
co-transfected
with
0.3
�g
pmCherry-HuR
and
0.3
�g
plasmid
encoding
EGFP-tagged
CVB3
2Apro,
2B,
2C,
3A,


3Cpro
2AD39E,2AL40F

3B,
,
3D,
VP1,
VP4-VP2-VP3,
,
2AS67F,2AY89L,2AY90L,2AV120M,2AG122E,or
2AD136N

;
EV71
2Apro;
or
the
empty
vector
pEGFP-C1.
Five
hours
later,
the
culture
media
were
removed
and
replaced
with
fresh
media.
Images
were
taken
using
an
Axiovert
200
fluorescence
microscope
(Carl
Zeiss,
Gottingen,
Germany)
at
24
h
post-transfection.
Some
cells
were
seeded
in
48well
plates
and
transfected
with
plasmid
encoding
EGFP-
tagged
CVB3
2Apro,2AG122E
or
3Cpro.
Control
cells
were
transfected
with
pEGFP-C1.
After
24
h
post-transfection,
the
cells
were
fixed
for
immunofluorescence
assay
of
HuR
and
G3BP1.
To
investigate
eIF4G
cleavage,
HeLa
cells
were
transfected
with
plasmid
encoding
EGFP-tagged


2Apro,2AD39E,2AL40F,2AS67F,2AY89L,2AY90L


CVB3
,


2AV120M,2AG122E
2AD136N


or
.
Cells
were
harvested
at
24
h
post-transfection.
Whole-cell
lysates
were
subjected
to
sodium
dodecyl
sulfate-polyacrylamide
gel
electrophoresis
(SDS-PAGE)
and
western
blotting
analysis.


Virus
infection


HeLaEGFP-TIA1
cells
were
mock-infected,
or
infected
with
CVB3
or
EV71
at
a
multiplication
of
infection
(MOI)
of


10.
At
3
h
p.i.,
HeLaEGFP-TIA1
cells
were
fixed
for
immunofluorescence
assays.
HeLa
cells,
co-transfected
with
pmCherry-HuR
and
pEGFP-C1,
or
pEGFP-eIF4G
or
pEGFP-eIF4GG681E,
were
mock-infected
or
infected
with
CVB3
(MOI
=
10).
At
3,
4,
or
6
h
p.i.,
the
cells
were
washed
once
with
phosphate-buffered
saline
(PBS),
and
processed
for
imaging
using
an
Axiovert
200
fluorescence
microscope.
Arsenite
(Ars)
treatment


Ars
has
been
widely
used
as
a
stimulator
of
SGs
[2].
To
induce
SGs,
cells
were
treated
with
sodium
arsenite



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2014,
11:192
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(NaArs)
(Sigma-Aldrich,
St.
Louis,
MO)
at
a
concentration
of
0.5
mM
in
growth
medium
for
30
min.


Immunofluorescence


Following
virus
infection,
Ars
treatment,
or
plasmid
transfection
as
described
above,
the
cells
were
washed
once
with
PBS
and
then
fixed
with
4%
paraformaldehyde
at
room
temperature
for
30
min.
The
cells
were
treated
with
0.25%
Triton
X-100
for
10
min
and
blocked
with
PBS
containing
1%
bovine
serum
albumin
(BSA)
for
30
min
at
room
temperature.
Cells
were
then
incubated
overnight
at
4�C
with
primary
antibody
diluted
in
blocking
buffer
and
with
secondary
antibody
for
2
h
at
room
temperature
in
the
dark.
G3BP1
was
detected
via
a
monoclonal
anti-G3BP1
antibody
(611126,
BD
Transduction
Labs,
San
Jose,
CA)
at
a
dilution
of
1:200
and
a
CF555-labeled
goat
anti-mouse
secondary
antibody
(20231,
Biotium,
Hayward,
CA)
at
a
dilution
of
1:1000.
HuR
was
detected
via
a
polyclonal
antibody
(11910-1-AP,
Proteintech
Group
Inc.,
Chicago,
IL)
at
a
dilution
of
1:200
and
a
CF555-labeled
anti-rabbit
secondary
antibody
(20232,
Biotium)
at
a
dilution
of
1:1000.
The
cells
were
then
washed
three
times
with
PBS
and
stained
with
Hoechst
33342
(0.4
�g/ml
in
PBS).
Images
were
taken
using
an
Axiovert
200
fluorescence
microscope.


Western
blotting


Proteins
were
extracted
from
the
treated
cells
using
Pierce
RIPA
Buffer
with
PMSF
cocktail.
Approximately
2
�gof
the
extracted
proteins
were
applied
to
SDS-PAGE.
The
separated
proteins
were
transferred
to
a
polyvinylidene
fluoride
(PVDF)
membrane
(0.45
�m,
Millipore,
Billerica,
MA),
which
was
blocked
with
5%
nonfat
milk
for
2
h
at
37�C
and
incubated
with
primary
antibody
overnight
at
4�C.
A
polyclonal
rabbit
anti-eIF4G
antibody
(15704-1-AP,
Proteintech
Group
Inc.)
was
1:1000
diluted
to
detect
eIF4G;
a
polyclonal
anti-�-actin
antibody
(sc-130301,
Santa
Cruz
Biotechnology,
SantaCruz,
CA)
was
1:1000
diluted
to
detect
�-actin.
After
a
standard
washing,
the
membrane
was
incubated
with
horse
radish
peroxidase
(HRP)-labeled
secondary
antibody
(Zhongshan
Goldenbridge
Biotech,
Beijing,
China)
for
1
h
at
room
temperature
and
washed
again.
The
blots
were
stained
using
Super
Signal
kit
(Pierce,
Rockford,
IL)
and
imaged
by
a
LAS4000
charge-
coupled
camera
(Fujifilm,
Tokyo,
Japan).
The
�-actin
was
employed
as
a
loading
control.


Additional
file



Additional
file
1:
Table
S1.
PCR
primers
for
the
construction
of
plasmids
pEGFP-C1,
pEGFP-TIA1,
pmCherry-HuR
,
pEGFP-eIF4G
and
pEGFP-eIF4GG681E
.
Table
S2.
PCR
primers
for
the
construction
of
plasmids
expressing
CVB3
protein
2B,
3A,
3B,
3C,
and
3D.
Table
S3.
PCR
primer
sequences
used
for
the
site-directed
mutagenesis
of
2Apro.
Table
S4.
The
eIF4G
cleavage
activity
obtained
with
different
variants
of
PV
2Apro
[23-25].
Figure
S1.
Expression
of
CVB3
2Apro
or
EV71
2Apro
in
Vero
cells
results
in
SG
formation.
Vero
cells



were
co-transfected
with
pmCherry-HuR
and
pEGFP-C1
or
pEGFP-CVB3
2A


or
pEGFP-EV71
2A.
Nuclei
were
identified
by
Hoechst
33342.
The
2A


expression
and
mCherry-HuR-positive
SGs
were
determined
using
a


fluorescence
microscope
(�
400)
at
24
h
post-transfection.



Competing
interests


The
authors
declare
that
they
have
no
competing
interests.


Authors�
contributions


SW
participated
in
the
design
of
the
study,
performed
plasmid
construction,
immunofluorescence
assays,
and
western
blotting
and
drafted
the
manuscript.
YW
coordinated
the
experimental
design
and
helped
edit
the
manuscript.
LL
performed
viral
infection
and
cell
transfection.
XS
participated
in
the
design
of
the
study.
TW
and
XZ
performed
arsenite
treatment.
LT,
YL
and
YC
performed
cell
transfection.
XL
and
FZ
helped
coordinate
the
experimental
design.
WZ
and
ZZ
made
substantial
contributions
to
the
experimental
design
and
coordination.
All
authors
read
and
approved
the
final
manuscript.


Acknowledgments


This
study
was
supported
by
the
Natural
Science
Foundation
of
China
(NSFC)
(81271825
to
Z.
Zhong,
31270198
to
W.
Zhao,
81101234
to
T.
Lei,
81101235
to
Y.
Wang,
and
31300144
to
T.
Wang).
We
thank
the
Heilongjiang
Provincial
Key
Laboratory
of
Pathogens
and
Immunity,
and
the
Heilongjiang
Provincial
Science
and
Technology
Innovation
Team
in
Higher
Education
Institutes
for
Infection,
Harbin
Medical
University,
Harbin
150081,
China
for
technical
support.


Author
details


1Department
of
Microbiology,
Harbin
Medical
University,
Harbin
150081,
China.
2Department
of
Cell
Biology,
Harbin
Medical
University,
Harbin
150081,
China.
3Division
of
Gastroenterology
and
Hepatology,
Department
of
Medicine,
University
of
Florida-Jacksonville,
Jacksonville,
FL
32206,
USA.


Received:
12
April
2014
Accepted:
26
October
2014



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doi:10.1186/s12985-014-0192-1
Cite
this
article
as:
Wu
et
al.:
Protease
2A
induces
stress
granule
formation
during
coxsackievirus
B3
and
enterovirus
71
infections.
Virology
Journal
2014
11:192.



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