Inhibition of Hepatitis C Virus Replication by IFN

TheJournalofImmunologyInhibitionofHepatitisCVirusReplicationbyIFN-MediatedISGylationofHCV-NS5AMin-JungKimandJoo-YeonYooISG15isaubiquitin-likemoleculewhoseexpressionisinducedbytypeIIFN(IFN-a/b)orinresponsetovirusorbacterialinfection.ISG15orconjugationofISG15totargetproteinswasreportedtoplaycriticalrolesintheregulationofantiviralresponses.IFNrestrictsreplicationofhepatitisCvirus(HCV).However,molecularmechanismofIFN-a/bthatinhibitsHCVreplicationisnotclearyet.Inthecurrentstudy,wedemonstratedthatreplicationofHCVwasinhibitedbyoverexpressionofISG15andISG15-conjugationenzymesintheHCVsubgenomicrepliconcells.AmongvariousnonstructuralproteinsofHCV,NS5AwasidentifiedasthesubstrateforISGylation.Furthermore,proteinstabilityofNS5AwasdecreasedbyoverexpressionofISG15orISG15-conjugatingenzymes.TheinhibitoryeffectofISG15orISGylationonNS5Awasefficientlyblockedbysub-stitutionoflysineat379residuetoargininewithintheC-terminalregion,suggestingthatISGylationdirectlycontrolsproteinstabilityofNS5A.Finally,theinhibitoryeffectofIFN-a/bonHCVreplicationwasfurtherenhancedbyISGylation,suggestingISG15asatherapeutictoolforcombinedtherapywithIFNagainstHCV.TheJournalofImmunology,2010,185:4311–4318.HepatitisCvirus(HCV)isanenvelopedRNAvirusthatbelongstothegenusHepacivirus,familyFlaviviridae(1).TheHCVgenomeisasingle-stranded,9.6-kb–longRNAmolecule.Itencodesa3000-aapolyproteinthatundergoesposttranslationalprocessingtoyieldatleast10functionallydis-tinctviralproteins:core,envelopeprotein1(E1),envelopeprotein2(E2),p7,andnonstructuralprotein(NS)2,NS3,NS4A,NS4B,NS5A,andNS5B(2–4).UnlikeotherHCVnonstructuralproteins,NS5Adoesnothaveenzymaticactivity.Instead,HCV-NS5Aphys-icallyinteractswithvariouscellularproteins,suchasp53,IFN-induced,dsRNA-activatedproteinkinase,oranoveltranscriptionfactorSNF2-relatedCBPactivatorprotein,toelicitmodulatorrolesincell-cycleregulation,cellulartransformation,andantiviralim-muneresponses(5–7).HCV-NS5Aisamembrane-associatedpro-teinthatusesitsN-terminalamphipathichelixdomainandisusuallyfoundintheHCVRNAreplicationcomplex(8,9).HCV-NS5AisindispensableforHCVRNAreplication,becauseHCVgenomereplicatesinthelipiddroplet-associatedmembraneoftheendo-plasmicreticulum(ER)(10).ChronicinfectionofHCVisaleadingcauseofliverdisease,includingchronichepatitis,livercirrhosis,andhepatocellularcar-cinoma(11).WiththeabsenceofeffectivevaccinestopreventHCVinfection,combinedtreatmentofpegylatedIFN-awithribavirinisthemostfavorablechoicefortherapy.However,resistancetoIFNisoftenobservedinpatientswithHCVinfection(12,13).In-terestingly,mutationsinHCV-NS5AwerereportedtocorrelatewithresponsivenesstoIFNtherapyinpatients,althoughitsprecisemechanismisnotconclusive(14).ToovercomeIFNresistanceinpatientswithHCVinfection,thedevelopmentofchemicalsthatinhibitHCV-specificenzymaticactivitiesorantisenseoligomersthatinhibitHCVreplicationhasbeenundertaken(15–17).Pathogen-associatedmolecularpatternsofreplicatingHCVRNAgenomes,suchas59-triphosphateRNAordsRNA,arerecognizedbytheproductofRIG-Iinthecytoplasm.Ittriggerstheactivationofintracellularsignalingpathwaysandtranscriptionfactors,suchasNF-kBandIFNregulatoryfactors(18–22).AnoutcomeoftheseeventsistheproductionoftypeIIFN(IFN-a/b)(23,24).SecretedIFN-a/binducestheactivationoftheIFN-stimulatedgenefactor3complex,whichtranslocatestothenucleus,whereitbindstotheIFN-stimulatedresponseelementregionsoftargetgenestodirecttheexpressionofIFN-stimulatedgenes(ISGs).AmongthegenesinducedbyIFN,IFN-induced,dsRNA-activatedproteinkinase,MxA,andISG15functionaseffectormoleculesinthehostcellresponsetoviralinfection(24).MultiplestudieshaveshownaclearabsenceorlowlevelofIFN-a/binthehepatocytesofpatientswithchronicHCVinfection.Inaddition,patientswithchronicHCVinfectionshownosignificantlevelofISGinductionupontreatmentwithIFN(25).Basedonthesefindings,ithasbeensuggestedthatHCVviralproteinsdisturbtheinnateimmuneresponseofthehostandthatalteredhepaticISGexpressionisassociatedwithliverpathology.ISG15isatypeIIFN-inducible,ubiquitin-likeprotein.MostcomponentsforISG15conjugation(ISGylation),includingUbelL(E1,ISG15-activatingenzyme),UbcH8(E2,ISG15-conjugatingen-zyme),andHERC(E3,ISG15ligase),arehighlyresponsivetoIFN-a/b–inducedsignaltransduction(26,27).ExpressionofISG15andtheconjugationofISG15totargetproteinsarestronglypromotedbyIFN-a/btreatment,dsRNA,andviralorbacterialinfection(28).ProductionofISG15orISGylationisbelievedtoplayanimportantroleinestablishingtheantiviralstateofaninfectedcell.ISG15-deficientmicearehighlysusceptibletoinfluenza,herpes,andSind-bisviralinfections(29),whereastheprotectiveroleofoverexpressedISG15inSindbisvirus-infectedIFN-a/bR-deficientmicehasbeenreported(30).ItwasalsoreportedthatISGylationofcellularproteinsDepartmentofLifeSciences,PohangUniversityofScienceandTechnology,Pohang,RepublicofKoreaReceivedforpublicationJanuary13,2010.AcceptedforpublicationJuly29,2010.ThisworkwassupportedbyGrantA080084fromtheKoreaHealthcareTechnologyR&DProject.AddresscorrespondenceandreprintrequeststoDr.Joo-YeonYoo,DepartmentofLifeSciences,PohangUniversityofScienceandTechnology,