Expression of an IRF-3 fusion protein and mouse estrogen receptor, inhibits hepatitis C viral replication in RIG-I-defic...

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Expression of an IRF-3 fusion protein and mouse estrogen receptor, inhibits hepatitis C viral replication in RIG-I-deficient Huh 7.5 cells
Series Title:
Virology Journal
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Mixed Material
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English
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Yao, Luyu
Yan, Xiaobo
Dong, Huija
Nelson, David R.
Liu, Chen
Li, Xiaoyu
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BioMed Central
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Abstract:
Interferon Regulatory Factor-3 (IRF-3) plays a central role in the induction of interferon (IFN) production and succeeding interferon-stimulated genes (ISG) expression en route for restraining hepatitis C virus (HCV) infection. Here, we established a stable Huh7.5-IRF3ER cell line expressing a fusion protein of IRF-3 and mouse estrogen receptor (ER) to examine IFN production and anti-HCV effects of IRF-3 in retinoic acid inducible-gene-I (RIG-I) deficient Huh 7.5 cells. Homodimerization of the IRF-3ER fusion protein was detected by Western blotting after treatment with the estrogen receptor agonist 4-hydrotamoxifen (4-HT) in Huh7.5-IRF3ER cells. Expression of IFN-a, IFN-b, and their inhibitory effects on HCV replication were demonstrated by real-time polymerase chain reaction (PCR). Peak expression of IFN-a and IFN-b was achieved 24-hours post 4-HT treatment, coinciding with the appearance of phosphorylated signal transducer and activator of transcription (STAT) proteins. Additionally, HCV viral replication declined in time-dependent fashion. In previous studies, a novel IFN-mediated pathway regulating expression of 1-8U and heterogeneous nuclear ribonucleoprotein M (hnRNP M) inhibited HCV internal ribosomal entry site (IRES)-dependent translation. When expression of ISGs such as 1-8U and hnRNP M were measured in 4-HT-treated Huh7.5-IRF3ER cells, both genes were positively regulated by activation of the IRF-3ER fusion protein. In conclusion, the anti-HCV effects of IRF-3ER homodimerization inhibited HCV RNA replication as well as HCV IRESdependent translation in Huh7.5-IRF3ER cells. The results of this study indicate that IRF-3ER homodimerization is a key step to restore IFN expression in Huh7.5-IRF3ER cells and in achieving its anti-HCV effects.
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Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository, IR@UF, (www.uflib.ufl.edu/ufir) at the time of funding. The Institutional Repository at the University of Florida (IR@UF) is the digital archive for the intellectual output of the University of Florida community, with research, news, outreach and educational materials
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Virology Journal 2011 8:445

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University of Florida
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University of Florida
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doi - 10.1186/1743-422X-8-445
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AA00007507:00001


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RESEARCH OpenAccessExpressionofanIRF-3fusionproteinandmouse estrogenreceptor,inhibitshepatitisCviral replicationinRIG-I-deficientHuh7.5cellsLuyuYao1,XiaoboYan4,HuijiaDong2,DavidRNelson3,ChenLiu2andXiaoyuLi1*AbstractInterferonRegulatoryFactor-3(IRF-3)playsacentralroleintheinductionofinterferon(IFN)productionand succeedinginterferon-stimulatedgenes(ISG)expressionenrouteforrestraininghepatitisCvirus(HCV)infection. Here,weestablishedastableHuh7.5-IRF3ERcelllineexpressingafusionproteinofIRF-3andmouseestrogen receptor(ER)toexamineIFNproductionandanti-HCVeffectsofIRF-3inretinoicacidinducible-gene-I(RIG-I) deficientHuh7.5cells.HomodimerizationoftheIRF-3ERfusionproteinwasdetectedbyWesternblottingafter treatmentwiththeestrogenreceptoragonist4-hydrotamoxifen(4-HT)inHuh7.5-IRF3ERcells.ExpressionofIFNa IFNb,andtheirinhibitoryeffectsonHCVreplicationweredemonstratedbyreal-timepolymerasechainreaction (PCR).PeakexpressionofIFN-a andIFNb wasachieved24-hourspost4-HTtreatment,coincidingwiththe appearanceofphosphorylatedsignaltransducerandactivatoroftranscription(STAT)proteins.Additionally,HCV viralreplicationdeclinedintime-dependentfashion.Inpreviousstudies,anovelIFN-mediatedpathwayregulating expressionof1-8UandheterogeneousnuclearribonucleoproteinM(hnRNPM)inhibitedHCVinternalribosomal entrysite(IRES)-dependenttranslation.WhenexpressionofISGssuchas1-8UandhnRNPMweremeasuredin4HT-treatedHuh7.5-IRF3ERcells,bothgeneswerepositivelyregulatedbyactivationoftheIRF-3ERfusionprotein.In conclusion,theanti-HCVeffectsofIRF-3ERhomodimerizationinhibitedHCVRNAreplicationaswellasHCVIRESdependenttranslationinHuh7.5-IRF3ERcells.TheresultsofthisstudyindicatethatIRF-3ERhomodimerizationisa keysteptorestoreIFNexpressioninHuh7.5-IRF3ERcellsandinachievingitsanti-HCVeffects.IntroductionHepatitisCvirusinfectioncauseschronicliverdiseases, cirrhosis,andhepaticcellularcarcinoma(HCC)with170 millionpeopleworldwideand4millionpeopleinthe UnitedStatesreportedlyin fected(CDC,1998).Inadditiontoitsglobalhealthproblem[1],futureprojections suggestthatHCVrelatedmortalitywillincrease2-3-fold overthenextdecade[2]withmorethan180billionUS dollarsestimatedtotalsocialeconomiccostintheUnited States[3].ThestandardtreatmentofchronicHCVis anti-viraltherapywithIFNandribavirin(RBV)butno HCVvaccineavailable.DespiteadditionalchemotherapeuticsisonhandfortreatmentofgenotypeIHCV patientsrecently,theanti-viralmechanismsofIFN-based therapiesarenotwelldefined,butmostlikelyinvolvethe activationofhostinnateimmunitytolimitHCV replication. Duringmicrobialinfection,therecognitionofmicrobial componentsismediatedbyhost-specificcellularpathogen-recognitionreceptors(PPRs).PPRsaremembersof thetoll-likereceptor(TLRs)familyandarelocalizedeither tocellularplasma(TLR4forlipopolysaccharide(LPS)and viralenvelops)orendosomalmembranes(TLR3for dsRNA,TLR7/8forssRNAandTLR9forDNA[4-7]). Conversely,intracellulardsRNAisalsorecognizedbythe RIG-IcytosolicRNAhelicaseormelanomadifferentiation associatedgene(MDA)-5[8].RIG-IRNAhelicasewas foundtobeanessentialmediatorofanti-HCVeffectsdue toitsbindingtoun-capped5 -endand3 -endHCV dsRNA,triggeringhostinnateimmunity[9]. IFNsbindtotheIFNa / b receptor(IFNAR)ineitheran autocrineand/orparacrinemannertoinitiateapositive feedbackloopthatresultsintheproductionofmoretypeI IFNs.IFNARstriggertheactivationoftheJAK/STAT *Correspondence:Xiaoyu.li@jax.ufl.edu1DivisionofGastroenterologyandHepatology,DepartmentofMedicine, UniversityofFlorida-Jacksonville,FL32206,USA FulllistofauthorinformationisavailableattheendofthearticleYao etal VirologyJournal 2011, 8 :445 http://www.virologyj.com/content/8/1/445 2011Yaoetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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pathwaytophosphorylatetheSTATproteins.TheSTAT transcriptionfactorsassociatedwithIRF-9toformaheterotrimericcomplex,IFN-stimulatedgenefactor3 (ISGF3),initiatingthetranscriptionofIFN-stimulated genes(ISGs)andinhibitingthedifferentstagesofvirus replicationandelicitingananti-viralstateinthehost [10,11].DuringHCVinfection,theseanti-viraleffects includetheinhibitoryeffectsonhostandHCVtranslation [12,13],regulationofcellularproliferationandapoptosis [14],regulationofadaptiveimmunity[15,16],andrecruitmentofNKcellstothesiteofinfection[17,18]toclear HCVinfectionbyinhibitingHCVgeneexpressionand HCVreplication.PatientswithclearedHCVinfection withoutIFN-basedtreatmentprovidesstrongevidencefor thehostinnateimmuneresponseduringacuteHCVinfection[19,20]. Inordertostudythedirectanti-HCVresponseofIRF-3 activation,aninducibleHuh7.5-IRF3ERcelllinewasestablishedinRIG-IdeficientHuh7.5cellsthatallowIRF-3 proteinhomodimerformationinacytokine/receptor-independentfashion.Huh7.5cellsareahighlyadaptedand poorlydifferentiatedhepatomacelllinethatlackstheability toproducedetectableinterferon-a / b wheninfectedwith HCVJFH-1virus[21].Therefore,Huh7.5-IRF3ERcellsis anadequatesystemtostudythedownstreammolecular eventsofIRF-3activationandestablishmentofasubsequentanti-HCVstatewithoutRIG-IactivationinHuh7.5 cells.MaterialsandMethodsPlasmidsAmammalianexpressionvector,pTIRF3ER,wasconstructedasafusionproteinoftheIRF3gene(51.6Kd) [22]andC-terminalsequencesofthemouseestrogen receptor(310a.a.)[23]inthepEF6/V5-HisTOPOTA vector(Invitrogen,Carlsbad,CA).TheplasmidpJFH-1 containsafull-lengthHCVgenomiccDNA[24].TheplasmidpRL-HLisadicistronicconstructthatmediatesCapdependentandHCVIRES-dependenttranslation[25]. Synthetic4-hydroxytamoxifen(4-HT)waspurchasedfrom Sigma(SaintLouis,MO)anddissolvedinethanolasa 5mMstocksolution.CelllinesHumanhepatomaHuh7.5cells[26]weregrowninDulbecco smodifiedEagle smedium(Invitrogen).Toestablish theHuh7.5-IRF3ERcellline,Huh7.5cellsweretransfectedwiththeplasmidpTIRF3ERandLipofectin(Invitrogen).Blasticidin(Invitrogen)(10 g/ml)wasusedforthe cloneselection24-hoursaftertransfection.Mediumwas changedevery3dayswithfreshBlasticidinuntilday14,at whichtime,positivecloneswerepropagated.Theclones wereamplifiedandIRF-3ERdimerformationwasmeasuredbyWesternblottingafter4-HTtreatment.DetectionofIRF-3ERdimers,p-STAT1(S727),p-STAT3 (Y705),and1-8UproteinbyWesternblottingHuh7.5-IRF3ERcellmonolayerswerewashedinphosphatebufferedsaline(PBS)post4-HTtreatmentwithproteaseinhibitorcocktail(Sigma).PreparationofHuh7.5IRF3ERcelllysateswasperformedasreportedpreviously [27].Cellularlysateswereseparatedbysodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS-PAGE)(6% gelforIRF-3ERdimers;8%gelforp-STAT1(S727)and p-STAT3(Y705)).Westernblottingwascarriedoutas previouslyreported[27]with antibodiesforactin(Santz CruzBiotechnology,Inc.,SantaCruz,CA),p-STAT3 (Y705)(CellSignaling,Bos ton,MA),p-STAT3(Y705) (CellSignaling),andSTAT1(SantzCruz).WesternblottingofSTAT1andSTAT3proteinswereperformedwith thesamePVDFmembraneusedfordetectionofp-STAT1 (S727)andp-STAT3(Y705)afterstrippingtheblot(Bioradstrippingbuffer).HCVJFH-1stocksandHCVinfectionPreparationandtitrationofHCVJFH-1viruswasreported previously[24].Forexamininganti-HCVeffects,Huh7.5IRF3ERcellswereincubatedwith0.5MOIJFH-1HCVfor 14daystoachievefullyinfectedHuh7.5-IRF3ERmonolayercells[28].TheHuh7.5-IRF3ERcellswerethentreatedwith4-HTfor72,48and24hourspriortocollecting totalcellularRNA.Huh7.5-IRF3ERcellswithout4-HT treatmentfor72hourswereusedascontrol.TotalRNA wasisolatedfordetectingHCVRNAbyreal-timePCR.DetectionofIFN-a andIFN-b inHuh7.5-IRF3ERcellsHuh7.5-IRF3ERcellsweretreatedwith4-HTfor72,48, and24hourspriortocollectingcellularlysates.Controlis Huh7.5-IRF3ERcellsthatdidnotreceive4-HTtreatment for72hours.TotalcellularRNAwasisolatedfordetecting IFNa orIFNb RNAbyreal-timePCR.Real-TimePCRassayTotalcellularRNAwasisolatedfrominfectedHuh7.5IRF3ERmonolayersbyTrizol(Invitrogen).First-strand cDNAweresynthesizedfrom1 gtotalcellularRNAby reversetranscription(20 lofreactionvolume).SuperscriptII(200Ureversetranscriptaseperreaction)anda RT-PCRkit(Invitrogen)wasusedtoprimewitholigo (dT)12-18forfirst-strandsynthesisaccordingtothemanufacturer sinstructions.Taqmanprimerswereobtained fromAppliedBioSystems.Reactionswereconductedina 96-wellMyiQcycler(Bio-Rad,Hercules,CA).FluorescencewasmonitoredduringeveryPCRcycleatthe annealingstep.TheprimersforHCVJFH-1are:forward, 5 -CGGAATTGCCGGGAAGAC-3 ;reverse,5 -CAAA TGGCCGGGCATAGAG-3 ;FAMprobe,5 -CTTTC TTGGATAAACCC-3 .TheprimersforIFNa are:forward,5 -GGGATGAGGACCTCCTAGACAAATT-3 ;Yao etal VirologyJournal 2011, 8 :445 http://www.virologyj.com/content/8/1/445 Page2of8

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reverse,5 -ACACAGGCTTCCAAGTCATTC-AG-3 ; FAMprobe,5 -CTGCACCGAACTCTAC-3 .TheprimersforIFNb are:forward,5 -TGGCTGGAATGAGACTATTGTTGAG-3 ;reverse,5 -CAGGACTGTCTTCA GATGG-TTTATCT-3 ;FAMprobe,5 -CCTCCTGGC TAATGTC-3 .GADPHprimerswerepurchasedfromthe AppliedBiosystems.PCRwasperformedwiththefollowingconditions:50C,2min;95C,10min;(95C,15s; 60C,1min)for40cycles.RelativeRNAlevelindicates statisticalquantificationofalteredRNAlevelsfromthese cellularlysateswithdifferentprimers.Sampleswererunin triplicateandtheresultswereanalyzedusingtheBio-Rad iQ5software;meansthestandarderrorofthemeanare shown.LuciferaseassaysHuh7.5-IRF3ERcellswereculturedin6-wellplatesand transfectedwiththeplasmidpRL-HLandlipofectamine 2000(Invitrogen).After24-hoursoftransfection,Huh7.5IRF3ERcellsweretreatedwith4-HTfor96,72,and 48hourspriortopreparingcelllysates.ControlHuh7.5IRF3ERcellswereincubatedfor96hoursintheabsence of4-HT.Allsampleswereanalyzedforluciferaseactivity usingtheDual-LuciferaseReporterAssaySystemKit(Promega,Madison,WI)intriplicate.Thetranslationefficiencywascalculatedasaproportionofcontrol(100%).StatisticalanalysisDifferentcellularlysateswerecollectedforanalysisof luciferaseactivityorrelativeRNAlevelfromHuh7.5IRF3ERcellswithspecialtreatment.Resultsofthese studiesareexpressedasmeansstandarddeviation (SD).ResultsDimerizationofIRF-3ERfusionproteininducedby4-HTin Huh7.5-IRF3ERcellsActivationofIRF-3orIRF-7isacriticalstepduringvirus infection,promotingthemostpotenttypeIIFNproduction.Previousstudiesshowedtheconstitutivelyactive forms(serinesreplacedbyphosphomimeticaspartate aminoacids)ofhumanIRF-3proteinexertstheabilityto modulatetheapoptoticandanti-tumorpropertiesafter beingdeliveredbyrecombinantadenovirusintomacrophages[22].Inourstudies,afusionproteinofIRF-3and C-terminalsequences(310a.a.)ofmouseestrogenreceptorwasusedtoestablishthestableHuh7.5-IRF3ERcell line.Inpreviousstudies,mouseestrogenreceptorwas effectiveatinducingdimerizationofSTAT1andSTAT3 fusionproteinsafter4-HTtreatment[27,23,29].Inthese studiesfromusandothers,4-HTwastitratedtoaconcentrationof1 Mthatachievedthehighestexpression ofSTAT1ERandSTAT3ERdimerizationandthestrongestinhibitoryeffectsonHCVRNAreplication[23,29]. Inourstudies,4-HT-treatmentalonewasalsodemonstratedtohavenoanti-HCVeffects[23].Inthisstudy, similarsequencesfromthemouseERC-terminaldomain werefusedtotheC-terminusoftheIRF-3gene.InFigure 1,Westernblottingwithanti-IRF-3antibodywasusedto detecttheIRF-3aswellastheIRF-3ERmonomerand dimerproteins.Lane1showsendogenousIRF3protein (56.1kd)butnoIRF-3ERfusionproteininHuh7.5cells treatedwith4-HT.Lane2showsbothIRF-3andIRF3ER(monomer)(90kd)inHuh7.5-IRF3ERcellswithout 4-HTtreatment.Lane3showsthat4-HTtreatmentof Huh7.5-IRF3ERcellsinducesIRF-3ERfusionprotein dimerformation(180kd)inadditiontoIRF-3protein andIRF-3ERmonomers.ThedensityofIRF-3ERdimers waslessthanthedensityofIRF-3ERmonomers(Figure 1,lane3),whichcouldbeexplainedbythedenaturing conditionsusedintheanalysisassuggestedinourpreviousreport,includingSDS-polyacrylamidegelelectrophoresis,RIPAlysisbuffer,andboilingduringWestern blotting[27].Interestingly,asmallamountofIRF-3ER dimerformationwasdetectedinHuh7.5-IRF3ERcells without4-HTtreatment(Figure1,lane2).Thismaybe dueeithertoauto-dimerizationofIRF-3ERordimerformationinducedbytraceestrogeninthetissueculture medium.MultipleformsoftheIRF-3ERfusionprotein werealsodetected(Figure1,lane3).Ourdataindicates theIRF-3ERfusionproteinapproachisaneffective meanstoachieveIRF-3homodimerizationwith4-HT treatment.ExpressionofIFNsafteractivationoftheIRF-3ERfusion proteinDuetodeficientRIG-IgenefunctioninHuh7.5cells, virusinfectionwillnotleadtoIRF-3activationandIFN Figure1 DimerizationoftheIRF-3ERfusionproteininHuh7.5IRF3ERcellline.Huh7.5-IRF3ERandHuh7.5cellsweretreatedwith orwithout4-HT.Totalcellularproteinwasextractedandanalyzed forWesternblottingwithanti-IRF3antibody.Lane1,Huh7.5cells treatedwith4-HT.Lane2,Huh7.5-IRF3ERcellswithout4-HT treatment.Lane3,Huh7.5-IRF3ERcellstreatedwith4-HT. Yao etal VirologyJournal 2011, 8 :445 http://www.virologyj.com/content/8/1/445 Page3of8

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secretion[21].ThisphenomenonallowsustostudyIRF-3 genefunctionagainstHCVinfectionbyestablishinga stableHuh7.5-IRF3ERcellline.FusionproteinsofSTAT1 andSTAT3withthemouseestrogenreceptorprovideda usefulmeanstostudydimerizationofthoseproteinsand resultinginanti-HCVstatus[27,23].Inthisstudy,the IRF-3genewasfusedwithsameC-terminalsequencesof mouseestrogenreceptorasreported[27,23]forinducing IRF-3ERdimerizationby4-H Ttreatment.Expressionof typeIIFNs( a and b )wasexaminedafter4-HTtreatment byreal-timePCR.InFigure2Aand2B,IFNa andIFNb increasedandpeaked24hoursafter4-HTinduction.To furtherdemonstratethebiologicalactivitiesofIFNa and IFNb afterIRF-3ERdimerization,Westernblottingwas usedtodetectphosphorylatedSTAT1andSTAT3.In Figure3A,phosphorylate dSTAT1wasdetectedwithan antibodyagainstSTAT1(S727)inHuh7.5andHuh7.5IRF3ERcells(Figure3A,lane1,2,3).Differentamounts ofphosphorylatedSTAT1wereobservedinbothHuh7.5 cellsandHuh7.5-IRF3ERcells(Figure3A,lane1and2). Therewerenoappreciabletime-dependentdifferencesin phosphorylatedSTAT1inHuh7.5-IRF3ERcellswithor without4-HTtreatment(Figure3A,lane2to9).This observationisconsistentwit htheauto-dimerizationof IRF-3ERfusionprotein(Figure1,lane2)toproduceIFNs (controlsofFigure2Aand2B).InFigure3B,phosphorylatedSTAT3wasexamined;therewasnodifference betweenHuh7.5,Huh7.5-IRF3ERcellswithorwithout4HTtreatment(Figure3B,lane1to9).Thisphenomenon couldbeexplainedbytheconstantactivationofIRF-7to induceexpressionofIFNa whichactivatesthetypeIIFN pathwaythroughSTAT3phosphorylation[10].Total STAT1andSTAT3proteinswasusedasinternalcontrols anddemonstratednodifferenceswithorwithout4-HT treatmentonHuh7.5-IRF3ERcellsorHuh7.5cells(Figure 3Aand3B,lane1to9).InhibitoryeffectsofIRF-3ERdimerizationonHCVJFH-1 virusreplicationHuh7.5-IRF3ERcellswerefurtherexaminedforitsinhibitoryeffectsonHCVJFH-1viralreplicationafter4-HT treatment.Huh7.5-IRF3ERcellswereinoculatedwith 0.5MOIofJFH-1virusstockandculturedfor14days toachievefullHCVJFH-1infectedHuh7.5-IRF3ERcell state.TheinfectedHuh7.5-IRF3ERcellsweretreated with4-HT(1 M)attheindicatedtimesandharvested atthelast-samplecollectionpointforanalysisofHCV RNAbyreal-timePCR.TheinfectedHuh7.5-IRF3ER cellswereusedascontrolwithout4-HTtreatmentfor 72hours.InFigure4A,HCVJFH-1replication decreasedto50%ofcontrolafter24hoursof4-HT treatment.ThisdataindicatesthatIRF-3ERdimerization after4-HTtreatmenthasinhibitoryeffectsonHCV JFH-1replicationandwascorrelatedwiththeproductionofIFNa andIFNb (Figure2Aand2B).Tofurther separateHCVJFH-1viralRNAreplicationandviral translation,theplasmidpRL-HL,containingCap-dependentRenillaluciferasetranslationandHCVIRESmediatedFireflyluciferasetranslationstartsites,was usedinthisstudy[25].AftertransfectionofpRL-HL, Huh7.5-IRF3ERcelllysateswereharvestedatvarious timesafter4-HTtreatmentforanalysisofluciferase activity.InFigure4B,bothCap-dependentandHCV IRES-mediatedtranslationwasreducedinHuh7.5IRF3ERcellsafter4-HTtreatmentinatime-dependent fashion.Thisdatashowsstrongevidencethatactivation oftheIRF-3ERfusionproteinnotonlyinhibitsJFH-1 viralRNAreplicationbutalsoinhibitsCap-dependent andHCVIRES-mediatedtranslation. Figure2 ExpressionofIFNa andIFNb inHuh7.5-IRF3ERcells with4-HTtreatment .Huh7.5-IRF3ERcellsweretreatedwith4-HT for72,48,and24hourspriortocollectingcellularlysates.Controlis Huh7.5-IRF3ERcellsthatdidnotreceive4-HTtreatmentfor72 hours.TotalcellularRNAwasisolatedfordetectingIFNa orIFNb RNAbyreal-timePCR. A .ExpressionofIFNa inHuh7.5-IRF3ERcells. B .ExpressionofIFNb inHuh7.5-IRF3ERcells. Yao etal VirologyJournal 2011, 8 :445 http://www.virologyj.com/content/8/1/445 Page4of8

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ExpressionofISGsinHuh7.5-IRF3ERcellsAlloftheIFNtypesactivateJAK/STATpathways,regulatingtheexpressionofover300ISGsinorderto achievetheiranti-viraleffects[30].Inourpreviousstudies,wedemonstratedanovelpathwaybywhichIFN inhibitsHCVIRES-mediatedtranslationthroughup-regulating1-8Ugeneexpression[27]anddown-regulating expressionofthehnRNPMgene(unpublisheddata).In thisstudy,wemeasured1-8UandhnRNPMexpression inHuh7.5-IRF3ERcellswithandwithout4-HTtreatment.InFigure5A,the1-8Uproteinwasdetectedby Westernblottingandwasup-regulatedinHuh7.5IRF3ERcellsafter4-HTtreatment.Duetoauto-dimerizationofIRF-3ERfusionproteininHuh7.5-IRF3ER cells,thefold-inductionof1-8Uprotein(Figure5A, lane1,2,and3)isnotasrobustasdescribedinour previousreportinwhichtheSTAT1genewasactivated [27].Real-timequantitative reverse-transcriptionPCR wasusedtodetectandmeasurehnRNPMmRNA expressioninHuh7.5-IRF3ERcells.After4-HTtreatment,hnRNPMmRNAlevelsweredown-regulatedina time-dependentfashion(Figure5B).Thisdataconfirms thatactivationoftheIRF-3ERfusionproteintriggersa cellularanti-HCVstatethroughinducingIFNsproductionandregulatingISGexpression.DiscussionHostimmunity,includinginnateimmunityandadaptive immunity,isanimportantandcomplicatedsystemdedicatedtothetaskofdefendingthehostfrommicrobial infectionandcancerdevelopment.Innateimmunityprovidesanimmediate(firstline)replytoamicrobialinfection,specificallyforviralinfections,whilealsocontrolling thelaterantigen-specificadaptiveresponse.Akeyaspect oftheantiviralinnateimmuneresponseisthesynthesis andsecretionoftypeIINFs( a and b ),whichexhibit Figure3 PhosophorylationofSTAT1andSTAT3proteinsin Huh7.5-IRF3ERcells .Huh7.5andHuh7.5-IRF3ERcellswereusedto detectphosophorylationofSTAT1andSTAT3proteins.Huh7.5IRF3ERcellsweretreatedwithoutorwith4-HTtoinduceIRF-3ER dimerization.TotalproteinwasextractedandanalyzedbyWestern blottingwithanti-p-STAT1(S727)antibody,anti-STAT1antibody ( FigureA );anti-p-STAT3(Y705)antibodyandanti-STAT3antibody ( FigureB ),respectively.Lane1,Huh7.5cellswith4-HT;lane2, Huh7.5-IRF3ERwithout4-HT;lane3tolane9,Huh7.5-IRF3ERwith4HTinductionfrom0.5to72hours. Figure4 Anti-HCVeffectsoftheIRF-3ERfusionproteinin Huh7.5-IRF3ERcells A InhibitoryeffectsofIRF-3ER dimerizationonHCVRNAreplication .Huh7.5-IRF3ERcellswere inoculatedwithHCVJFH-1stockataMOI0.5for14daysandthen 4-HT(1 M)wasaddedat72,48,and24hourspriortoend-point forsamplecollection.ControlindicatestheJFH-1infectedHuh7.5STAT1ERcellswithout4-HTtreatmentfor72hours.HCVJFH-1RNA levelsweremeasuredbyquantitativereal-timePCRintriplicate. RelativeJFH-1RNAlevelwascalculatedasproportionofcontrol(1.0). ThedataispresentedafternormalizationwithaninternalGAPDH control.Theerrorbarsindicatethevariationpresentinthree independentassays. B.InhibitoryeffectsofIRF-3ERdimerization onHCVIRES-mediatedtranslation .Huh7.5-IRF3ERcellswere transfectedwiththeplasmidpRL-HL.After24hoursofincubationat 37Cand5%CO2,transfectedHuh7.5-STAT1ERcellsweretreated with4-HTfor48,72,and96hoursforanalysesofluciferaseactivity. ControlHuh7.5-IRF3ERcellsreceivedthepRL-HLcDNAplasmidbut didnotreceive4-HTtreatment.Translationefficiencyforeach samplewascalculatedasproportionofcontrol(100%).Theerror barsindicatethevariationofthreeindependentassays. Yao etal VirologyJournal 2011, 8 :445 http://www.virologyj.com/content/8/1/445 Page5of8

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antiviral,anti-proliferative,andimmunomodulatoryfunctions.Twokeystepsarerequiredtoelicitaneffective antiviralinnateimmuneresponse:a.detectionofthe invadingvirusbyimmunesystemreceptors;b.initiation ofproteinsignalingcascadesthatregulatethesynthesis ofIFNs.Virusesarehighlyinfectiouspathogensthat dependonhostcellularmachineryforsurvivalandreplication.Mostviralinfections,likethecommoncold causedbyRhinoviruses,areefficientlyresolvedbythe hostinnateandadaptiveimmunesystem.Forotherviral infections,suchaschronichepatitisBorCviralinfection, thehostinnateandadaptiveimmunityresponseisunable toclearthemeffectivelya ndtheybecomepersistent infections.SeveralfamiliesofPPRshavebeendemonstratedtoinspectthecellu larmicro-environmentfor microbialinfectiontotargetthepathogen-associated molecularpatterns(PAMPs),aconservedstructuralmoietyessentialformicrobialsurvival.Toll-likereceptors (TLRs3,4,7,8,and9)inadditiontoRIG-Iaremajor PPRsthatrecognizedifferenttypesofvirally-derived nucleicacidsorintracellulardsRNAtoinitiatesignaling cascadesleadingtoproductionoftypeIIFNs(detailsin reviews[11,31,32]).Themechanismsbywhichdifferent virusesinduceauniqueIFN-mediatedantiviralresponse appeartorequireselectiveactivationofmembersofthe IRFfamilyofproteins(IRF-1toIRF-9).Thusfar,IRF-3 andIRF-7havebeenshowntobemajorregulatorsof IFNgeneexpression[33,10]. ThetypeIIFNs,represent edbymultiplesubtypesof IFNa inadditiontoonesubtypeIFNb ,arekeycytokinesinthisprocess,mountinganimmediateantiviral responseaswellasadaptiveimmunity.IFN-mediated anti-viraleffectsarecarriedoutusingdifferentmechanismsthataredependentonthetypeofviralinfection,but theseanti-viraleffectsarealldependentonIRF-3activation[35,34,33,21,7].ActivationofIRF-3proteinsappears torecruittheTankBindingKinase1(TBK1)andinhibitorofI B-relatedkinaseepsilon(IKK )[7]throughtheir interactionwiththeRIG-IRNAhelicase[36],resultingin phosphorylationofIRF-3,itsdimerization,nucleartranslocation,andtranscriptional activationthroughbinding toIFN-stimulatedresponseelements(ISREs)[37].ActivatedIRF-3interactswithnuclearfactorB(NFB)and transcriptionalfactor-2/c-Juntoformatranscriptionally activeenhanceosomecomplexon IFNA1 and IFNB gene promoters. Inourstudies,weutilizedanIRF-3/mouseERfusion proteinexpressingplasmidinordertoachieveIRF-3ER activationinacytokine/rece ptor-independentfashion. Ourresultsdemonstrated thatIRF-3ERhomodimers (Figure1,lane3)triggeredthedownstreampathwaysto produceIFNa andIFNb (Figure2Aand2B).TheantiHCVeffects,inducedby4-HTinHuh7.5-IRF3ERcells, wereachievedbydecreasingHCVRNAreplicationand HCVIRES-mediatedtranslation.Thisisconsistentwith ourpreviousstudieswhichachievedactivationofSTAT1/ andSTAT3/mouseERfusionproteins.Activationofthe IRF-3ERfusionproteinby4-HTtreatmentprovides strongevidencethatthisisnecessaryandsufficientto increaseIFNa andIFNb expressioninHuh7.5-IRF3ER cells(Figure2Aand2B).OurdatashowingthatIRF-3ER activationtriggersthedownstreampathway,activatingthe JAK/STATpathwayandregulatingISGexpression.Detectionofp-STAT1(S727)andp-STAT3(Y705)inHuh7.5IRF3ERcellsprovidesastrongevidenceforactivationof Jak/STATspathwaybyIFNs(Figure3Aand3B).Although Figure5 Expressionof1-8UandhnRNPMgenesinHuh7.5IRF3ERcells A Geneexpressionof1-8UinHuh7.5-IRF3ERcells Huh7.5-IRF3ERcelllysateswereharvestedat0,24,and48hours afteradding4-HTtoinduceIRF-3ERdimerization.Totalproteinwas extractedandanalyzedbyWesternblottingwithananti-1-8U antibody.Lane1,Huh7.5-IRF-3ERcelllysatefromcellstreatedwith 4-HTat0hours;lane2and3,Huh7.5-STAT1ERcelllysatesfromcells treatedwith4-HTfor24and48hours.Actinproteinwasusedasan internalcontrol. B ExpressionofhnRNPMinHuh7.5-IRF3ER cells .Huh7.5-IRF3ERcellsweretreatedby4-HT(1 M)for72,48, and24hourspriortocollectingcelllysates.Controlismock treatmentof4-HTfor72hours.ExpressionofhnRNPMandGAPDH (usedasinternalcontrol)mRNAwasmeasuredusingTaqman primersfromAppliedBiosystemsintriplicate.RelativehnRNPM RNAlevelswerecalculatedasaproportionofthecontrol(1.0). Yao etal VirologyJournal 2011, 8 :445 http://www.virologyj.com/content/8/1/445 Page6of8

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themechanismofIFNactionagainstHCVreplicationhas notbeenwelldefined,recentstudiessuggestthatIFNs haveagreatimpactonHCVreplicationbyinterrupting HCVIRES-mediatedtranslation[38,12].ClinicaldataconfirmedthesefindingsinastudyofHCVIRES-mediated translationinchronicHCVpatientsreceivingIFNtreatment,inwhichtheefficiencyofHCVIRES-mediated translationwasreducedinIFN-treatedHCVpatients [39,40].Inourstudy,theinhibitoryeffectsofHCVRNA replicationandHCVIRES-mediatedtranslationwereconfirmedinHuh7.5-IRF3ERcellsaftertreatmentwith4-HT (Figure4Aand4B). Here,wepresentdatademonstratingthatactivationof theIRF-3generestoresIFNproductioninRIG-IdeficientHuh7.5cells.Theanti-HCVeffectswereachieved inHuh7.5-IRF3ERcellsbydecreasingbothHCVRNA replicationandHCVIRESmediatedtranslation. Recently,twonewgenes,1-8UandhnRNPM,wereisolatedinourstudiesduetotheirabilitytomodulatecellularCap-dependentandHCVIRES-mediated translationandregulatedbySTAT1pathwayactivation.Acknowledgements XiaoyuLiissupportedbytheJuniorFacultyStart-upFundingfromthe UniversityofFlorida-Jacksonville.DavidNelsonwassupportedbyNIH UL1RR029890-0,R01AI061158,andNIH-NCIK24CA139570-0.Theauthors appreciateDr.TakajiWakitawhoprovidedtheHCVJFH-1plasmid,Dr. CharlesRicewhoprovidedtheHuh7.5cells,Dr.JohnHiscottwho providedfulllengthIRF-3cDNAclone,andDr.StanleyLemonwho provideduswiththepRL-HLplasmid.Authorsappreciatetheproofreading andsuggestionsforthismanuscriptbyDr.MichaelHaas(Universityof Florida-Jacksonville). ThisworksupportedinpartbytheNIH/NCRRClinicalandTranslational ScienceAwardtotheUniversityofFloridaUL1RR029890.Publicationofthis articlewasfundedinpartbytheUniversityofFloridaOpen-Access PublishingFund. 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