TRIM25 Enhances the Antiviral Action of Zinc-Finger Antiviral Protein

25 Pages·2017·5.06 MB·English

RESEARCHARTICLE
TRIM25 Enhances the Antiviral Action of Zinc-
Finger Antiviral Protein (ZAP)
MelodyM.H.Li1,ZerlinaLau2,PamelaCheung2,EduardoG.Aguilar1,William
M.Schneider1,LeoniaBozzacco1,HenrikMolina3,EugenBuehler4,AkinoriTakaoka5,
CharlesM.Rice1,DanP.Felsenfeld2¤,MargaretR.MacDonald1*
1 LaboratoryofVirologyandInfectiousDisease,TheRockefellerUniversity,NewYork,NewYork,United
StatesofAmerica,2 IntegratedScreeningCore,ExperimentalTherapeuticsInstitute,IcahnSchoolof
MedicineatMountSinai,NewYork,NewYork,UnitedStatesofAmerica,3 ProteomicsResourceCenter,
TheRockefellerUniversity,NewYork,NewYork,UnitedStatesofAmerica,4 Trans-NIHRNAiScreening
a1111111111
Facility,DivisionofPreclinicalInnovation,NationalCenterforAdvancingTranslationalSciences,NIH,
a1111111111 Rockville,Maryland,UnitedStatesofAmerica,5 DivisionofSignalinginCancerandImmunology,Institutefor
a1111111111 GeneticMedicine,HokkaidoUniversity,Sapporo,Japan
a1111111111
a1111111111 ¤ Currentaddress:CHDIFoundation/CHDIManagement,Inc,NewYork,NewYork,UnitedStatesof
America
*[email protected]
Abstract
OPENACCESS
Citation:LiMMH,LauZ,CheungP,AguilarEG, Thehostfactorandinterferon(IFN)-stimulatedgene(ISG)product,zinc-fingerantiviralpro-
SchneiderWM,BozzaccoL,etal.(2017)TRIM25
tein(ZAP),inhibitsanumberofdiversevirusesbyusurpingandintersectingwithmultiple
EnhancestheAntiviralActionofZinc-Finger
AntiviralProtein(ZAP).PLoSPathog13(1): cellularpathways.Toelucidateitsantiviralmechanism,weperformaloss-of-function
e1006145.doi:10.1371/journal.ppat.1006145 genome-wideRNAiscreentoidentifycellularcofactorsrequiredforZAPantiviralactivity
Editor:AnaFernandez-Sesma,IcahnSchoolof againsttheprototypealphavirus,Sindbisvirus(SINV).Inordertoexcludeoff-targeteffects,
MedicineatMountSinai,UNITEDSTATES wecarryoutstringentconfirmatoryassaystoverifythetophits.ImportantZAP-liaisingpart-
Received:June1,2016 nersidentifiedincludeproteinsinvolvedinmembraneionpermeability,typeIIFNsignaling,
andpost-translationalproteinmodification.Thefactorcontributingmosttotheantiviralfunc-
Accepted:December20,2016
tionofZAPisTRIM25,anE3ubiquitinandISG15ligase.Wedemonstrateherethat
Published:January6,2017
TRIM25interactswithZAPthroughtheSPRYdomain,andTRIM25mutantslackingthe
Copyright:Thisisanopenaccessarticle,freeofall RINGorcoiledcoildomainfailtostimulateZAP’santiviralactivity,suggestingthatboth
copyright,andmaybefreelyreproduced,
TRIM25ligaseactivityanditsabilitytoformoligomersarecriticalforitscofactorfunction.
distributed,transmitted,modified,builtupon,or
otherwiseusedbyanyoneforanylawfulpurpose. TRIM25increasesthemodificationofboththeshortandlongZAPisoformsbyK48-and
TheworkismadeavailableundertheCreative K63-linkedpolyubiquitin,althoughubiquitinationofZAPdoesnotdirectlyaffectitsantiviral
CommonsCC0publicdomaindedication.
activity.However,TRIM25iscriticalforZAP’sabilitytoinhibittranslationoftheincoming
DataAvailabilityStatement:Allrelevantdataare SINVgenome.Takentogether,thesedatauncoverTRIM25asabonafideZAPcofactor
withinthepaperanditsSupportingInformation
thatleadstoincreasedZAPmodificationenhancingitstranslationalinhibitionactivity.
files.
Funding:Theworkwassupportedinpartbythe
Women&SciencePostdoctoralFellowship,the
StarrFoundation,GreenbergMedicalResearch
Institute,NationalInstitutesofHealth(NIH)grants AuthorSummary
AI097089,AI114873,andAI091707,and
Organismshaveevolvedvariousinnatestrategiestodefendagainstpathogens.During
anonymousdonors.TheuseoftheBDLSRIIwas
virusinfection,thecellsensestheviralnucleicacidandproducestypeIinterferon,which
supportedbyGrant#UL1TR000043fromthe
NationalCenterforAdvancingTranslational alertstheneighboringcells.SignalingoftypeIinterferontriggersexpressionofawide
Sciences(NCATS)andNationalInstitutesofHealth
PLOSPathogens|DOI:10.1371/journal.ppat.1006145 January6,2017 1/25
TRIM25IsaZAPCofactor
(NIH)ClinicalandTranslationalScienceAward
(CTSA)program.Thefundershadnoroleinstudy arrayofgenes,someofwhichencodeproteinsthatinhibitthereplicationofdiversevirus
design,datacollectionandanalysis,decisionto families.Itisnotclearwhatdeterminesthebroadyetspecificactivityoftheseinterferon-
publish,orpreparationofthemanuscript.
inducedgeneproducts.Identificationofcellularcofactorsandpathwaysrequiredforthe
CompetingInterests:Theauthorshavedeclared functionofthesebroadlyantiviralproteinswouldhelpelucidatetheirmechanisms.Here,
thatnocompetinginterestsexist. weperformagenomewideknockdownscreentoidentifyhostfactorsimportantforthe
antiviralactionofzincfingerantiviralprotein(ZAP),abroad-spectruminhibitoryprotein
inducedbyinterferon.WefindthatZAPsynergizeswithhostproteinswithdivergent
functionsandidentifyTRIM25asabonafideZAPcofactor.TRIM25bindstobothsplice
isoformsofZAP,andstimulatestheirubiquitinationandfunctionbyfacilitatingtheabil-
ityofZAPtoblockviraltranslation.Ourdatashedslightontheantiviralmechanismof
ZAPandadvancesourunderstandingofhostfactorcontributionstoinnateimmune
responsesagainstviralinfections.
Introduction
Therecentre-emergenceandspreadofvirusesbeyondtheirnormalgeographicdistribution
haveaffectedcountriesworldwide.Understandingthebiologyofhostfactorswithbroadanti-
viralactivityiscrucialtovaccineanddrugdevelopmenteffortstocounteractexistingand
emergingviralinfections.
Asafirstlineofdefenseagainstviruses,thehostproducestypeIinterferons(IFNs),which
signalthroughtheJAK/STATpathwaytoinducehundredsofIFN-stimulatedgenes(ISGs)
thatblockvariousstepsofthevirallifecycle(reviewedin[1]).AmongtheISGs,zincfinger
antiviralprotein(ZAP),encodedbytheZC3HAV1 gene,inhibitsalphaviruses,filoviruses,hep-
atitisBvirus,retroviruses,andtheLINE-1andAluretroelements[2–8].However,ZAPdoes
notinhibityellowfevervirus,vesicularstomatitisvirus,andherpessimplexvirus1(HSV-1)
[3].ItisnotwellunderstoodwhatdeterminesthebroadyetspecificantiviralactivityofZAP.
ZAP,alsocalledPARP13,isamemberofthepoly(ADP-ribose)polymerase(PARP)family
andisalternativelyspliced.ThelongisoformofZAP(ZAPL)containsaPARP-likedomainon
theC-terminusthatismissingintheshortisoform(ZAPS).ThisPARP-likedomainisnot
enzymaticallyactive[9],althoughexchangeoftheinactivecatalytictriadinZAPLtothatof
theactivePARPscompletelyabolishesitsantiviralactivity[10],suggestinganimportantyet
unknownroleofthePARP-likedomainintheantiviralfunctionofZAP.Severalstudieshave
demonstrateddistinctactivitiesforthetwoisoforms.ZAPLismoreactiveagainstalphaviruses,
suchasSINVandSemlikiForestvirus,thanZAPS,andcarriessignaturesofpositiveselection
[11,12].WhilebothisoformsareinducedbyIFN,ZAPSisupregulatedmorethanZAPLby
virusinfectionandtypeIIFN[5,13,14].
DiversecellularpathwayshavebeenimplicatedinZAP’sfunction(reviewedin[15]),butits
precisemechanismisunknown.ItispossiblethatZAPinteractswithmultiplehostfactors,
andtheinvolvementofthosefactorsinthevirallifecycleiswhatprovidesthespecificity.For
example,ZAPbindsRNAandrecruitstheexosomecomplextotargetviralRNAsfordegrada-
tion[5–7,16–18].ZAPalsodirectlyinhibitstranslationoftheincomingalphaviralgenome
[3],withinterferenceintheinteractionbetweeneIF4AandeIF4G[19]implicatedasone
mechanism.Inaddition,ZAPsynergizeswithotherISGsforitsmaximalactivityandupregu-
latesRIG-I-mediatedIFN-βproduction[14,20].ThesestudiessupportamodelinwhichZAP
interactswithvarioushostfactorsandcellularcomplexestoachieveanoptimalantiviralstate
againstdiverseviruses.
PLOSPathogens|DOI:10.1371/journal.ppat.1006145 January6,2017 2/25
TRIM25IsaZAPCofactor
InanattempttounifythedivergentpathwaysinwhichZAPisinvolvedandtouncover
novelcofactorsthatareimportantforZAP’sinhibitoryactivity,weperformedagenome-wide
siRNAscreeninacelllineinducibleforZAPexpression.Large-scaleRNAiscreensallowusto
takeanunbiasedapproachtointerrogateeverygeneinthegenome.However,off-targeteffects
leadtofalsepositivehitsandseverelylimitthevalueofgenome-widescreens[21,22].To
addressthisweperformedarigoroussetofconfirmatoryassaystoverifythetophitsand
excludeoff-targeteffects.WeidentifiedseveralgenesthatsynergizewithZAPtotargetSINV
orinhibitSINVindependentlyofZAP.Amongthehits,TRIM25wasvalidatedtobeacofactor
ofZAP.TRIM25isanE3ubiquitinandISG15ligase,andisresponsibleforthepolyubiquitina-
tionandactivationofRIG-I[23–25].WegeneratedCRISPRclonesinZC3HAV1-knockout
293TcellswhereTRIM25expressionissignificantlyreducedandfurtherconfirmedthat
TRIM25synergizeswithbothZAPSandZAPLtoblockSINVreplication.Ourdatademon-
stratesthatTRIM25triggersubiquitinationofZAPandenhancesitsantiviralactivitythrough
inhibitionofviraltranslation,highlightingtheimportanceofcofactorsinthemechanismsof
broadlyantiviralproteins.
Results
Agenome-widesiRNAscreenrevealsnovelhostfactorsthatsynergize
withZAPorexhibitZAP-independentantiviralactivity
Weperformedagenome-widescreenwithpooledsiRNAsfromDharmacontoidentifygenes
thatarerequiredforZAP’santiviralactivity(seeFig1Aforscreenworkflow).Viralreplication
islowinT-REx-hZAPcellswhenZAPexpressionisinduced,andsilencingofZC3HAV1
increasesreplicationofaluciferase-encodingSINV,Toto1101/Luc,by2logs.Thepremiseof
thescreenisasfollows:shouldknockdownofanessentialcofactorrenderZAPnonfunctional,
viralreplicationwillberestored,resultinginincreasedluciferaseactivity(referto“ZAPcofac-
torsiRNA”columninFig1A).Thescreenwasperformedintriplicatetoimproverobustness,
andweidentified480genes,whosesilencingsignificantlyelevatedSINVToto1101/Lucrepli-
cationwithanaveragerobustZscoreof>3(Fig1B).Asexpected,ZC3HAV1 wasthetophit
withanaveragerobustZscoreof582.65;thiswasfollowedbyBAI3(165.56),TRIM25(116.52)
andRICS(100.42)(seeS1Tablefortheentireresults).
Normalizedpercentactivation(NPA)androbustZscorewereutilizedforhitselection
[26].ThegeneswiththehighestNPAandrobustZscore>3inallthreereplicatewellswere
chosenforvalidationinasecondaryscreen(91genes).SincesiRNAscanactasmicroRNAs
andtargetgenesnon-specificallythroughtheirseedsequences[27],weincluded4genesthat
werepotentialoff-targetcandidatesfromHaystackanalysis(PDIK1L,SNAP25,FOXK1,
DGAT2L3).Inaddition,weincluded1genebasedonoverlapwithISGsthatwepreviously
foundassynergisticwithZAPinanoverexpressionscreen(MAP3K14;[20]),and6genesfrom
pathwaysthatweresignificantlyenrichedbutwerenotonthetop91list(APC,FZD2,GFRA1,
JAK1,SP1,andWNT8B).
Were-screenedthecandidategeneswithalibraryofsinglesiRNAsobtainedfromadif-
ferentcompany(Ambion)toexcludehitsthataremediatedbyoff-targeteffectsfromfurther
characterization.Knockdownof5genesby6.25nMsiRNA(Fig2A)andknockdownof13
genesby25nMsiRNA(Fig2B)significantlyincreasedSINVToto1101/Lucreplication(see
S2Tablefortheentireresults).Amongthem,ZC3HAV1 wasidentifiedasthetophit.
TRIM25,KCNH5,GCS1andJAK1werealsohitsatbothsiRNAconcentrations.Inaddition
totheT-REx-hZAPcellsusedfortheprimaryscreen,a293clonethatisinducibleforthe
expressionofratZAPC88Rmutant,adominantnegativeinhibitorofZAPfunction[28],
wasalsotestedinparallel.SinceendogenouslyexpressedZAPisantiviral,thiscellline
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TRIM25IsaZAPCofactor
Fig1.Aloss-of-functionRNAiscreenuncoversmanygenesthatsignificantlyreducetheantiviral
activityofZAPwhensilenced.(A)Theexperimentaloutlineofthegenome-widesiRNAscreenisshown.
T-REx-hZAPcellstransfectedwithcontrolorgene-specificsiRNAweretreatedwithdoxycyclinetoinduce
ZAPSoverexpressiononedaypost-transfectionandinfectedwithSINVToto1101/Luctwodayspost-
transfection.Celllysateswereharvestedformeasurementofluciferaseactivityat24hpost-infection(p.i.).
RelativeluciferaseunitsrepresentthelevelofSINVreplication.Cellstreatedwiththecontrolnon-targeting
(NT)pooledsiRNAhavelowSINVreplicationwhileZAPknockdownbyZC3HAV1-specificpooledsiRNA
rescuesviralreplicationby2logs.Thelargedynamicrangeinwhichhypotheticalhits(ZAPcofactors)were
identifiedisplottedontherightsideofthegraph.(B)PooledsiRNAstargetingtheentirehumangenome
(Dharmacon)weretestedintriplicateandgeneswithanaveragerobustZscoreofgreaterthan3areplotted.
ZC3HAV1ishighlightedinredwhilethetophitsimmediatelyfollowingZC3HAV1arehighlightedinblue.
doi:10.1371/journal.ppat.1006145.g001
inhibitedforZAPfunctionallowedustoidentifyhitswithaZAP-independentantiviralrole.
SilencingofGCS1andGPRC5Dby6.25nM(Fig2C)and25nM(Fig2D)siRNAssignifi-
cantlyincreasedSINVToto1101/LucreplicationinthesecellswhereZAPisnotfunctional
(seeS2Tablefortheentireresults),suggestingthattheymightinhibitSINVinaZAP-inde-
pendentmanner.
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TRIM25IsaZAPCofactor
Fig2.SecondaryscreenconfirmstheZAP-dependentand-independentantiviraleffectsofhits.AcustomizedlibraryofindividualsiRNAs
(Ambion)targeting102hitsfromtheprimaryscreenwastestedintriplicateintwodifferentcelllines.DistributionofaverageZscoresforeachof
the3siRNAstargetingacandidategeneinthesecondaryscreenisshownhere.Thescreenwasperformedwith(A)6.25nMor(B)25nM
individualsiRNAsin293cellsinducedtoexpressZAPS(T-REx-hZAP),andwith(C)6.25nMor(D)25nMindividualsiRNAsin293cellsinduced
toexpresstherZAPC88Rdominantnegativemutant(T-REx-rZAPC88R).EachdotrepresentstheaverageZscoreofanindividualsiRNAtested
intriplicate.SilencedgeneswithanaverageZscoreof>3foratleast2outof3siRNAsareidentifiedandthesiRNAsarelabeledincolor.(Cand
D)TheaverageZscoresofTRIM25andZC3HAV1arealsoplottedtoindicatethatTRIM25doesnothaveZAP-independentantiviraleffects.
doi:10.1371/journal.ppat.1006145.g002
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TRIM25IsaZAPCofactor
TRIM25isabonafidecofactorofZAP
Next,wevalidatedthecandidateZAPcofactorsinalowerthroughputassay.Silencingof
TRIM25,KCNH5,JAK1,andZC3HAV1 ledtoincreasedvirusreplicationforatleast2ofthe3
siRNAs(Fig3A),whichwasconsistentwithreducedproteinlevelsofTRIM25(S1AFig)and
ZAP(S1BFig),andreducedmRNAlevelsofKCNH5andJAK1(S1CFig).Amongthecandi-
dates,3independentsiRNAstargetingTRIM25significantlyincreasedSINVToto1101/Luc
Fig3.TRIM25synergizeswithZAPtoblockSINVreplication.(A)CandidategenesthatsignificantlyincreasedSINVreplicationinthesecondary
screenwhensilencedbyindividualsiRNAsatbothconcentrationswerevalidatedinalargerscale24-wellplateformat.TriplicatewellsofT-REx-
hZAPcellsweretransfectedwiththeindicatedsiRNA,inducedtoexpressZAPS,andinfectedwithSINVToto1101/LucataMOIof10.Eachsymbol
representsthevalueobtainedfromasinglewellafter24hofinfection.WhitecirclesrepresentresultsusingpooledsiRNAcontrolsthatwereeitherNT
orZC3HAV1-specific.Thedataisrepresentativeof3independentexperiments.Asterisksindicatestatisticallysignificantdifferences(Student’st-test,
**,p<0.005;***,p<0.0005;****,p<0.0001).(B)TriplicatewellsofT-REx-hZAPcellsweretransfectedwiththeindicatedsiRNA,inducedtoexpress
ZAPS,andinfectedwithSINVToto1101/LucataMOIof10.ProteinexpressionlevelsofTRIM25andZAPforthesametransfectionsinaduplicate
wellweredeterminedbyimmunoblotting.β-actinwasusedasaloadingcontrol.Thedataisrepresentativeof4independentexperiments.Thep-
valuefromStudent’st-testisshown.(C)SINVreplicationininfected293TcellsinwhichZC3HAV1(left)orTRIM25(middle)weresilenced,andin
ZC3HAV1-null293TcellsinwhichTRIM25wassilenced(right)isplotted.At48hpost-transfectionwithsiRNA,cellswereinfectedwithSINV
Toto1101/LucataMOIof0.01,andlysedat6,12,24,and40hp.i.formeasurementofluciferaseactivity.Thedataisrepresentativeof3independent
experiments.Asterisksindicatestatisticallysignificantdifferences(two-wayANOVA,*,p<0.05;**,p<0.01;****,p<0.0001).
doi:10.1371/journal.ppat.1006145.g003
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TRIM25IsaZAPCofactor
replicationby10-to26-foldcomparedtotheNTcontrol(Fig3A).SinceTRIM25-targeting
siRNA#3wasmostefficientatrescuingSINVToto1101/Lucreplication,wedesignedand
testedanadditionalC911control.The9thto11thnucleotidesofthesiRNAweremutatedto
theircomplementarysequence,hencethedesignationC911,toruleoutthepossibilitythatthe
knockdownphenotypewasduetotheoff-targeteffectsofthesiRNA[29,30].TheC911con-
trolshouldloseitssiRNAactivitybutstillmaintainitsoff-targeteffectsasamicroRNA.While
TRIM25-targetingsiRNA#3rescuedSINVToto1101/Lucreplicationbyabout1logcompared
totheNTcontrol,#3-C911didnotleadtoincreasedviralreplicationanddidnotreducethe
proteinlevelofTRIM25(Fig3B),suggestingthatthephenotypeofsiRNA#3isduetospecific
silencingofTRIM25andnotinhibitionofanoff-targetgene.Furthermore,TRIM25was
silencedinZC3HAV1-knockout 293Tcells[14],whichweretheninfectedbySINVToto1101/
LuctodeterminewhetherendogenouslyexpressedTRIM25hasaZAP-independentantiviral
role.Ascontrols,ZC3HAV1 andTRIM25weresilencedin293Tcells.WhileZC3HAV1 (Fig
3C;left)andTRIM25(Fig3C;middle)silencingin293TcellsrestoredSINVToto1101/Luc
replicationby1–3logsby40hp.i.,silencingofTRIM25inZC3HAV1-knockout cellsdidnot
furtherincreaseSINVToto1101/LucreplicationcomparedtotheNTcontrol(Fig3C;right).
ThesedatasuggestthatTRIM25requiresZAPforitsanti-SINVactivity.
TRIM25interactswithZAPthroughitsSPRYdomain
Next,weaskedwhetherTRIM25physicallyinteractswithZAP.Weinfected293Twithendog-
enousTRIM25andZAPexpressionwithSINV,andimmunoprecipitatedTRIM25tolookfor
ZAPassociationatvarioustimepointsfollowinginfection.WefoundthatendogenousZAP
co-immunoprecipitatedwithendogenousTRIM25overthecourseofSINVinfection(Fig4A).
TherewerelessTRIM25andZAPproteinspresentat24hp.i.,whichisconsistentwiththe
cytopathiceffectsobservedatthattimepoint,leadingtolessTRIM25andZAPpulldown.Pre-
viously,TRIM25wasfoundtointeractwithZAPinthepresenceofRNAalthoughonlyZAPL
wasinvestigated[8].TodeterminetheinteractionofTRIM25anddifferentZAPisoforms,
ZAPSorZAPL,and/orTRIM25wereco-expressedinZC3HAV1-knockout 293Tcells,which
wereharvestedforco-immunoprecipitation.ImmunoprecipitationofbothZAPSandZAPL
byamonoclonalantibodyrecognizingtheN-terminalportionofhumanZAP(NZAP)pulled
downTRIM25,althoughmoreTRIM25isassociatedwithZAPL(Fig4B).TRIM25wasdra-
maticallymodifiedinthepresenceofZAPS,evidentbythepresenceinthewholecelllysates
(WCL)ofaladderofbandslargerthanthemolecularweightofTRIM25(Fig4B;WCL).
TRIM25consistsofaRINGdomain,twoBboxdomains,acoiledcoil(CCD)domain,anda
SPRYdomain.TheRINGdomainencodestheubiquitinligaseactivitywhiletheCCDdomain
isrequiredforoligomerizationofTRIMproteinsandtheSPRYdomainisimportantformedi-
atingproteininteractionsandsubstratespecificity[31].Next,weaskedwhichdomainin
TRIM25wasresponsibleforinteractionwithZAP.SincemoreZAPLassociateswithTRIM25,
weco-expressedsimilarlevelsofindividualTRIM25domainswithZAPLandimmunoprecipi-
tatedZAPL.WefoundthattheSPRYdomainofTRIM25butnotitsRINGandBbox/CCD
domainsco-immunoprecipitatedwithZAPL(Fig4C).ThesedatasuggestthatbothZAPiso-
formsformacomplexwithTRIM25,likelythroughinteractionwiththeSPRYdomainof
TRIM25.
BoththeE3ubiquitinligaseactivityandoligomerizationofTRIM25are
requiredforitsfunctionalinteractionwithZAP
SinceTRIM25isanE3ubiquitinligaseandhasbeenshowntobeimportantforubiquitinating
RIG-IandupregulatingRIG-I-mediatedIFN-βproduction,wehypothesizedthatTRIM25
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TRIM25IsaZAPCofactor
Fig4.TheSPRYdomainofTRIM25associateswithZAP.(A)293TcellswereinfectedwiththeSINVstrainToto1101(MOI=10),andlysates
wereharvestedat0,6,12and24hp.i.forco-immunoprecipitationwithananti-TRIM25antibodyandimmunoblotting.Thedataisrepresentative
of2independentexperiments.(B)HumanZAPshort(S)andlong(L)isoformsareshown,withtheCCCHfingers,TIPARP,WWEandPARP-like
domainsindicated.LightgreenshadingindicatessequencessharedbythetwoisoformswhereasthehatchedregioncontainingthePARP-like
domainisuniquetotheLisoform.WCLofZC3HAV1-knockout293TcellstransfectedwithV5-taggedTRIM25and/orZAPSorZAPLwereused
forco-immunoprecipitationwithananti-NZAPantibodyandimmunoblotting.(C)Full-length(FL)TRIM25isshown,withtheRING,Bbox,CCD,
andSPRYdomainsindicated.WCLofZC3HAV1-knockout293TcellstransfectedwithV5-taggedFLortruncatedTRIM25(RING,Bbox/CCD,
SPRYonly)andZAPLwereusedforco-immunoprecipitationwithananti-NZAPantibodyandimmunoblotting.DifferentamountsofTRIM25
domain-expressingconstructswereusedfortransfectioninordertoachievesimilarRING,Bbox/CCD,andSPRYexpression.Theasterisk
indicatesanon-specificband.(BandC)Thedataisrepresentativeof3independentexperiments.
doi:10.1371/journal.ppat.1006145.g004
alsoubiquitinatesZAPand/orotherproteinsthatcomplexwithZAPinordertostimulate
ZAP’santiviralactivity.Totestthis,wetargetedTRIM25byCRISPRinZC3HAV1-knockout
293TcellstointerrogatethefunctionalinteractionbetweenZAPisoformsandTRIM25.
CRISPRtargetingledtoeitherin-framedeletionsinallthreechromosomalcopiesofTRIM25
(cloneD)orframeshiftinsertionsintwochromosomalcopiesandanin-framedeletioninone
(cloneF),consistentwiththealmostundetectableproteinexpressionofTRIM25(S2Fig).
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TRIM25IsaZAPCofactor
Fig5.CRISPRtargetingofTRIM25leadstoincreasedvirusreplicationandboththeRINGandCCDdomainsofTRIM25arerequiredfor
ZAPactivation.(A)Wildtype(cloneE)andTRIM25loZC3HAV1-knockout293Tcells(clonesDandF)weretransfectedwithemptyvectororvector
expressingZAPSorZAPLandinfectedwithToto1101/Luc(MOI=0.01)2dayspost-transfection.(B)TRIM25loZC3HAV1-knockout293Tcells
(clonesDandF)werereconstitutedwithexpressionofFLortruncatedTRIM25(ΔRING,ΔCCD)and/orZAPSorZAPL,andinfectedwithToto1101/
Luc(MOI=10)2dayspost-transfection.(AandB)Thedataisrepresentativeof2independentexperimentsperformedonbothclonesDandF.Cell
lysateswereharvestedformeasurementofluciferaseactivityat24hp.i.RelativeluciferaseunitsrepresentthelevelofSINVreplication.Asterisks
indicatestatisticallysignificantdifferences(Student’st-test,*,p<0.05;**,p<0.005;***,p<0.0005).
doi:10.1371/journal.ppat.1006145.g005
ClonesDandFaredesignatedTRIM25lo.CloneEiswildtypeandhassimilarTRIM25protein
expressionastheparentalZC3HAV1-knockout 293Tcells(S2Fig).BothTRIM25loclones(D
andF)weretestedinthesubsequentexperimentsandshowedsimilarresults.Wetransfected
TRIM25locellswithZAP-expressingconstructsandinfectedthemwithSINVToto1101/Luc.
TRIM25knockdownsignificantlyenhancedSINVToto1101/Lucreplicationinthepresence
ofZAPSandZAPLoverexpression(Fig5A).Furthermore,weco-transfectedTRIM25locells
withTRIM25-andZAP-expressingconstructstodeterminetheantiviraleffectofthedifferent
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TRIM25IsaZAPCofactor
TRIM25andZAPcombinations.FLTRIM25aloneinhibitedSINVreplication,likelydueto
overexpression(Fig5B).WefoundthatFLTRIM25enhancedtheactivityofZAPSmorethan
theRING-andCCD-deficientTRIM25mutantsatbothhighMOI(Fig5B)andlowMOI
(S3AFig),inthecontextofsimilarexpressionlevelsofFLandmutantTRIM25proteins(S3B
Fig).ThisdatasuggeststhatboththeE3ligaseactivityandoligomerizationofTRIM25are
importantfortheactivationofZAP.However,FLTRIM25doesnotsignificantlyenhance
SINVinhibitionbyZAPL(Fig5BandS3AFig),whichhintsatpotentialisoform-specificdif-
ferencesintheZAP-TRIM25synergy.
TRIM25mediatesubiquitinationofZAPSandZAPL
SincetheE3ligase-defectiveTRIM25(ΔRING)failedtostimulatetheactivityofZAPS(Fig
5B),wehypothesizedthatTRIM25mightactbyubiquitinatingZAPand/orotherhostpro-
teins.First,weaskedwhetherZAPisubiquitinated.293Tcellstransfectedwithaconstruct
expressingHA-taggedubiquitinwerelysedunderdenaturingconditionstodisruptprotein-
proteininteractions,andendogenousZAPwasimmunoprecipitatedwithananti-ZAPpoly-
clonalantibodyandprobedwithananti-HAantibody.Acontrolanti-GFPantibodyofthe
samespeciesastheanti-ZAPantibodywasusedtocheckfornon-specificpulldown.We
foundthatZAPwasmodifiedbyubiquitinationatbaselineanduponSINVinfection(Fig
6A).WhenZAPSandZAPLwereoverexpressedintheZC3HAV1-knockout 293Tcellsin
thepresenceofHA-taggedubiquitinandsubjecttoimmunoprecipitation,bothZAPiso-
formswerefoundtobemodifiedbyubiquitin,suggestingthatthemodifiedlysine(s)are
likelysharedbybothisoformsandlocatedinZAPS(Fig6B).However,ZAPLismorepolyu-
biquitinatedthanZAPS,whichhintstoadditionalubiquitinationsitesinthePARP-like
domain.Inaddition,weaskedwhetherTRIM25isimplicatedintheubiquitinationofZAP
anddeterminedtheeffectofbothendogenousandoverexpressedTRIM25onZAPubiquiti-
nationlevel.BothZAPSandZAPLubiquitinationintheTRIM25locellswasreducedcom-
paredtothatintheparentalTRIM25sufficientZC3HAV1-knockout cells(Fig6C).
Consistentwiththat,overexpressedTRIM25dramaticallyincreasedthelevelofmodifica-
tionofZAPisoformsbybothendogenous(Fig6D)andoverexpressedubiquitin(Fig6E).
Furthermore,wedeterminedwhichlinkagetypeofpolyubiquitinTRIM25inducesonZAP
byoverexpressingubiquitinmutants,inwhichallthelysineresiduesaremutatedexceptfor
K48orK63.WeimmunoprecipitatedZAPisoformsthatwereco-expressedwithTRIM25,
andHA-taggedwildtypeormutant(K48,K63)ubiquitin,andfoundthatbothZAPSand
ZAPLwereubiquitinatedbybothK48-andK63-linkedpolyubiquitin(Fig6F).Together,
thesedatasuggestthatTRIM25isresponsibleforZAPmodification.
Next,thelysineresiduesinZAPSthatwerepredictedtobeubiquitinatedwithmediumto
highconfidencebyUbPredandCKSAAP_UbSitewerechangedtoarginineresiduesindividu-
ally(K226R,K296R,K314R,K401R,K416R,K448R,andK629R)orincombination(K296R/
K448R,7UbΔ)todeterminewhetherpotentialubiquitinationatanyofthesesitesaffectsZAP’s
antiviralactivity.Moreover,apreviousstudy,inwhichaglobalapproachwastakentoidentify
alltheubiquitinatedproteinsinthecell,reportedatrypticpeptidewithaubiquitinatedlysine
inZAPL(EEGK (glygly)LLFYATSR)[32].Weconfirmedthatthisresidueisubiquitinated
783
usingatargetedmassspectrometryapproachandthelysinewasmutatedtoarginineinZAPL
(K783R).Whenweco-expressedthepanelofZAPubiquitinationsitemutantswithTRIM25
andimmunoprecipitatedZAPtodeterminethelevelofmodification,wefoundthatthemuta-
tionsdiminishedthelevelofZAPubiquitinationtovariousdegrees(S4AFig).Mostimpor-
tantly,introductionofall7mutationsatthesametimealmostcompletelyabrogatedZAP
ubiquitination(referto“S7UbΔ”inS4AFig).However,theZAPS7UbΔmutantwasstill
PLOSPathogens|DOI:10.1371/journal.ppat.1006145 January6,2017 10/25