Infectious pancreatic necrosis virus proteins VP2, VP3, VP4 and VP5 antagonize IFNa1 promoter activation while VP1 induces IFNa1

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Virus Research

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / l o c a t e / v i r u s r e s

Infectious pancreatic necrosis virus proteins VP2, VP3, VP4 and VP5 antagonize IFNa1 promoter activation while VP1 induces IFNa1

Silje Lauksund, Linn Greiner-Tollersrud, Chia-Jung Chang, Børre Robertsen

^∗

NorwegianCollegeofFisheryScience,UniversityofTromsø,9037Tromsø,Norway

a r t i c l e i n f o

Articlehistory:

Received10September2014 Receivedinrevisedform 11November2014 Accepted13November2014 Availableonline24November2014

Keywords:

IPNV Interferon Antagonism IPS-1 IRF1 IRF3

a b s t ra c t

Infectiouspancreaticnecrosisvirus(IPNV)isoneofthemajorviralpathogenscausingdiseaseinfarmed Atlanticsalmonworldwide.InthepresentworkweshowthatseveraloftheIPNproteinshavepowerful antagonisticpropertiesagainsttypeIIFNinductioninAtlanticsalmon.EachoftheﬁveIPNVgenescloned intoanexpressionvectorweretestedfortheabilitytoinﬂuenceactivationoftheAtlanticsalmonIFNa1 promoterbytheinterferonpromoterinducingproteinone(IPS-1)orinterferonregulatoryfactors(IRF).

ThisshowedthatpreVP2,VP3andVP5inhibitedactivationofbothpromoters,whileVP4onlyantagonized activationoftheIFNa1promoter.TheviralproteaseVP4wasthemostpotentinhibitorofIFNinduction, apparentlytargetingtheIRF1andIRF3branchofthesignalingcascade.VP4antagonismisindependent ofitsproteaseactivitysincethecatalyticallydeadmutantVP4K674AinhibitedactivationoftheIFNa1 promotertoasimilarextentaswildtypeVP4.IncontrasttotheotherIPNVproteins,theRNA-dependent RNApolymeraseVP1activatedtheIFNa1promoter.TheabilitytoactivatetheIFNresponsewasdisrupted inthemutantVP1S163A,whichhaslosttheabilitytoproducedsRNA.VP1alsoexhibitedsynergistic effectswithIRF1andIRF3ininducinganIFNa1-dependentantiviralstateincells.Takentogetherthese resultssuggestthatIPNVhasdevelopedmultipleIFNantagonisticpropertiestopreventIFN-induction byVP1anditsdsRNAgenome.

1. Introduction

Infectiouspancreaticnecrosisvirus(IPNV)causesdiseaseand mortalityinhatchery-rearedsalmonidfryandinAtlanticsalmon postsmolts after transfer to the sea (Hill and Way, 1995; Jarp etal.,1995;Smailetal.,1992).ElucidationofhowIPNVinteracts with the innate immune system of Atlantic salmon is impor- tant to understand its pathogenic properties. IPNV belongs to thegenusAquabirnavirusoftheBirnaviridaefamily.Thegenome consistsoftwodouble-stranded(ds)RNAsegments,packedina non-envelopedsingle-shelledicosahedricalcapsid(Coulibalyetal., 2005;Dobos,1976; Pousetal., 2005).SegmentBencodesVP1, whichisa94kDaRNAdependentRNApolymerase(RdRp)found both in a free and a genome-linkedform inthe virion(Dobos, 1995).SegmentAcontainsasinglelargeopenreadingframe(ORF) thatencodesapolyprotein,whichisco-translationallycleavedby thenon-structuralproteaseVP4togeneratethematurestructural proteinsVP2andVP3(Duncanetal.,1987).VP2istheoutercap- sidproteinwhile VP3 isfoundin theinner surfaceof theviral

∗Correspondingauthor.Tel.:+4777644487;fax:+4777646020.

E-mailaddress:[email protected](B.Robertsen).

capsid.VP3hasrecentlybeenshowntointeractwithVP1andwith double-strandedRNA(Pedersenetal.,2007).Inaddition,segment AcontainsasmalloverlappingORFencodingVP5,whichisanon- structuralproteinwithundeﬁnedproperties,proposedtohavean anti-apoptoticfunctionandtobeofimportanceforvirulence(Santi etal.,2005).ThesizeofVP5variesdependingontheisolate,from 15kDato3.3kDa,butsomeisolateslacktheVP5reading-frame altogether(Heppelletal.,1995;Skjesoletal.,2011).

Toestablishasystemicinfection,viruseshavedevelopedawide rangeofmechanismstoevadeandsubvertthetypeIinterferon (IFN)system,which playsa crucialrolein theinnate immunity againstvirusesofvertebrates(vandenBroeketal.,1995).Type I IFNs are induced upon recognition of viral RNA by the RNA helicasesRIG-IandMDA5inthecytoplasm,andbythetoll-like receptorsTLR3andTLR7,whichareembeddedinthemembraneof endosomes(ArpaiaandBarton,2011;TakeuchiandAkira,2007).

Upon release, IFNsinduce a range ofantiviral proteinsboth in non-infectedandinfectedcellspreventingfurthervirusinfection.

AmongtheantiviralproteinsareMx,ISG15,viperinandPKR(Chin and Cresswell, 2001; Liu et al., 2011;Samuel, 2001).Targeting initiationoftranscription ofIFNandIFN-induced genesarefre- quentlyusedstrategiesforvirusestoestablishaninfectioninthe host(RandallandGoodbourn,2008).IPNVVP4andVP5havebeen http://dx.doi.org/10.1016/j.virusres.2014.11.018

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showntoinhibitIFN-inducedactivationoftheMxpromoter,but theeffectofIPNVproteinsoninductionoftypeIIFNisunknown (Skjesoletal.,2009).

AtlanticsalmonpossessesatleastfourtypeIIFNsubtypes,IFNa, IFNb, IFNcand IFNd,which show largedifferencesin sequence andexpressionproperties(Svingerudetal.,2012).IFNa1,IFNband IFNcallshowpotentantiviralactivityagainstIPNVandtheyinduce Mxprotein,whichinhibitsIPNVreplication(Rokenesetal.,2007;

Svingerudetal.,2012).Inthisworkwechosetostudytheinﬂuence ofIPNVproteinsonactivationoftheAtlanticsalmonIFNa1pro- motersinceIFNa1isinducedinmostcellsuponstimulationwith dsRNA,whileIFNbandIFNcaremainlyinducedinlymphoidorgans (Sun et al.,2009; Svingerud et al., 2012). SalmonIFNa1 shows expressionpropertiesstrikinglysimilartomammalianIFN␤,which isinducedthroughtheRIG-I/MDA5and TLR3pathways(Bergan etal.,2006; Sunet al.,2009;Svingerudetal.,2012).Similarto thehumanIFN␤promoter,thesalmonIFNa1promoter hasone NF␬B-bindingsiteandtwoIRFbindingsites(Berganetal.,2006).

Teleost ﬁsh possess all key components of the RIG-I/MDA5 signalingpathway,includingRIG-I,MDA5,IPS-1,MITA,TBK1and IRFs(Berganetal.,2010;Biacchesietal.,2009;Changetal.,2011;

Fengetal.,2011;Hollandetal.,2008;Lauksundetal.,2009;Ohtani etal.,2011,2012;Simoraetal.,2010;Suetal.,2010;Sunetal., 2011; Zouet al., 2009). In mammals,RIG-I and MDA5 interact withtheadaptorproteinIPS-1uponbindingviralRNA(Berkeand Modis,2012;Jiangetal.,2011),whichactivatesthetranscription factorsIRF-3,IRF-7andNF␬B,resultinginstartofIFN␤transcription(RandallandGoodbourn,2008).InAtlanticsalmon,IPS-1,IRF1 andIRF3arestronginducersoftheIFNa1promoter(Berganetal., 2010;Lauksundetal.,2009).

In apreviouswork,we noticedthatIPNV infectionfailed to inducetheAtlanticsalmonIFNa1promoter(Berganetal.,2006).

Toelucidatethereasonwhythevirusdoesnotinduceasuccess- fulIFNresponse,wehereinvestigatedtheeffectoftheindividual IPNVproteinsonIFNa1induction.Thepresentworkdemonstrates thatIPNVhasdevelopedmultiplemechanismstoinhibitinduction ofIFNa1transcription.Surprisingly,however,wefoundthatVP1 stronglyactivatedIFNa1transcription,whichmayinpartexplain whythevirusneedspotentIFN-antagonisticproperties.

2. Materialsandmethods 2.1. Cellsandviruses

AtlanticsalmonTOcells(WergelandandJakobsen,2001)were obtained from Dr. Heidrunn Wergeland (University of Bergen, Bergen,Norway).TOcellswerecultivatedat20^◦CinL-15medium (Gibco, Life technologies) supplemented with1% non-essential aminoacids,100␮g/mlstreptomycin,100units/mlpenicillin(Life technologies)and8%fetalbovineserum(FBS)superior(Biochrom AG).IPNVserotypeSpN1(Christieetal.,1988)waspropagatedin CHSEcellsandviraltiterwasdeterminedtobe1×10⁷inCHSEcells basedontheTCID50method(42).Theviruswasstoredat−80^◦C untiluse(ReedandMuench,1938).

2.2. InfectionofTOcellsbyIPNV

TOcellswereseededin24-wellplatesand100%conﬂuentcells wereinfectedwith10MOIofIPNVorleftuntreated(control).The cells(treatedintriplicates)wereharvestedafter6,12,24and48h.

2.3. Neontransfectionsandluciferaseassays

TOcellsweresplit2:3thedaybeforetransfection.Thecellswere transfectedat80%conﬂuenceusingthe10␮lNeontransfection system(Lifetechnologies)withbufferRandatapulsevoltageof

Table1

Plasmidconstructs.

Plasmid Reference

pDESTmycVPl Skjesoletal.(2009)

pDESTmycpreVP2 Skjesoletal.(2009)

pDESTmycVP3 Skjesoletal.(2009)

pIPS-1 Lauksundetal.(2009)

pIRFIHA Berganetal.(2010)

pIRF3HA Berganetal.(2010)

pA1(-202) Berganetal.(2006)

pDESTmycVP1S163A Thisstudy

pDESTmycVP4K674A Thisstudy

pVP1ﬂag Thisstudy

pVP4ﬂag Thisstudy

pVP1S163Aﬂag Thisstudy

pVP4K674Aﬂag Thisstudy

pGL3basic Promegacorporation

pGL74 Promegacorporation

pcDNA3.1 Invitrogen–Lifetechnologies

pcDNA3.3 Invitrogen–Lifetechnologies

110,pulsewidth30ms,2pulses.Foreachtransfection450,000cells wereused.Foreachwellatotalof500ngluciferasevectorandtest vectorswereused,and50ngofthepGL74Renillaluciferasevector wasincludedasatransfectioncontrol.Theexpressionvectorsused areshowninTable1.Aftertransfectionthecellswereseededin24- wellplateswithL-15mediumwith12%FBS,withoutantibiotics, andincubatedat20^◦C.Cellswereharvestedin50␮lpassivelysis bufferand10␮lofthesamplewasmeasuredaccordingtothedual- luciferasereporterassaysystemprotocol(Promega).Resultsare shownasrelativelightunits(RLU)oftestreporter(ﬁreﬂyluciferase) overcontrolreporter(Renillaluciferase).

2.4. AntiviralassayandIFNneutralization

TOcellsweretransfectedin10␮lNeontransfectionreactions asdescribedintheprevioussection,resuspendedin400␮lgrowth mediawithoutantibioticsandtransferredto4wellsina96-well platecontaining100␮lgrowthmediawithoutantibiotics.Con- trolcellsweretreatedthesamewaywithoutplasmidpresent.The cellswereincubatedat20^◦Cfor72hpriortoinfection.Cellswere infectedwithIPNVatamultiplicityofinfection(MOI)of0.1inL- 15withoutsupplementsfor1hbeforechangingtogrowthmedia with2%FBS.Thesupernatantsfromthetransfectedcellswerehar- vested and stored at−80^◦C until useinantiviral and antibody neutralizationassay.FortheIFNneutralizationassay,TOcellswere seededin96wellplatesatadensityof2×10⁴cellsperwell.The supernatantwasdividedintwo,andonepartwaspre-incubated withIFNa1antibodyata1:100dilutionfor1hat37^◦C.Untreated supernatantwasalsoincubatedfor1hat37^◦C.Thecellswereincu- batedwith100␮lofeitheruntreatedorneutralizedsupernatant for24h,andsubsequentlyinfectedwithIPNVataMOIof0.1as previouslydescribed.Whencompletecytopathogeniceffect(CPE) wasobservedincontrolcellsfourdaysafterinfection,cellsurvival wasdeterminedbycrystalvioletstainingasdescribedin(Lauksund etal.,2009).ViraltitrationwasdeterminedinTOcellsseededin96 wellplatesatadensityof2×10⁴cellsperwell.Calculationofviral titerwasdeterminedbytheTCID50 method(ReedandMuench, 1938).

2.5. Site-directedmutagenesis

Mutations were introduced to the VP1 and VP4 genes by site-directedmutagenesisusingtheGeneArt^®Site-DirectedMuta- genesisSystemfromInvitrogenandthefollowingprimers:

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VP1S163Afwd:5-CTGCAATACGGGTCCGGCGCCTACTCAGGACAA- CTC-3 and VP1S163Arev:5-GAGTTGTCCTGAGTAGGCGCCGGA- CCCGTATTGCAG-3;

VP4K674Afwd: 5-GCGGTGTAGACATCGCAGCCATCGCAGCCCAT- GAAC-3andVP4K674Arev:5-GTTCATGGGCTGCGATGGCTGCGA- TGTCTACACCGC-3.

Mutagenesiswasperformedaccordingtothespeciﬁcationsin thekit.

2.6. Subcloning

Plasmidscontaining theVP1,VP1S163A,VP4 and VP4K674A genesequenceswereusedastemplatesandnewconstructswith FlagtagwereampliﬁedbyPCRwithprimerswhichincludedaFlag tagsequence(DYKDDDDK)intheN-terminalofeachORFsegment.

ThesePCRproductswereclonedintoapcDNA3.3plasmid(Invitro- gen).Thefollowingprimersequenceswereused:

VP1fwd, 5-CACCATGGACTACAAAGACGATGACGACAAGATGTCG- GACATCTTCAATTC-3andVP1rev,5-TCAGTTTCTTCTCTGCTTCTC- CCGACG-3;

VP4fwd,5-CACCATGGACTACAAAGACGATGACGACAAGAGCGGA- GGGCCCGACGGAAA-3andVP4rev,5-TCATGCATTTGATGCCATC- AGCTCTCCCAGGTACT-3.

2.7. RelativequantitativerealtimePCR(qPCR)analysis

RNAwasisolatedfromneon-transfectedorinfectedTO-cellsby usinganRNeasyMiniKit(Qiagen,SantaClarita,CA).RNAintegrity was veriﬁed by 1% agar gel electrophoresis, Quantity of RNA wasassessedwiththeNanoDrop^® ND-1000spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Criterion to includeRNAsampleswas260/280nmabsorbanceratioof1.9–2.2.

0.2␮g of DNase-treated total RNA was reverse-transcribed to cDNA with oligo(dT) primers in a 20␮l reaction (QuantiTect^® ReverseTranscriptionKit(QIAGEN)).ForPCRprimerefficiency,a ten-folddilutionserieswitheightmeasuringpointsfromarandom poolof cDNAwasused.Theefficiencieswerecalculated bythe formula:efficiency(%)=(10⁽⁻^1/slope)−1)*100.PCRprimerefficien- cies(between2.04and2.22)werecloseto100%allowinguseof the 2⁻CT methodfor calculationof relative gene expression (43).TheqPCRassayswerecarriedoutwiththeABIPRISM7700 Sequence DetectionSystemusing2×Fast SYBR^® Green Master Mix (AppliedBiosystems)in a 15␮lreactionvolume,with6␮l 1:10dilutedcDNA,andprimer concentrationsof0.50␮M.PCRs wererunintriplicatesin96-wellopticalplatessystemunderthe following conditions:95^◦Cfor 5min (pre-incubation),95^◦C for 5s,60^◦Cfor15s,72^◦Cfor15s(45cycles)andcontinuousincrease from65^◦Cto97^◦Cwithstandardramprate(meltingcurve).18S used as a reference gene showed stable expression in control and test samples according to the BestKeeper software (Pfaffl et al., 2002).The following primerswere used for the relative quantification: IFNa, 5-TGCAGTATGCAGAGCGTGTG-3 and 5- TCTCCTCCCATCTGGTCCAG-3;IFNb,5-TGCATTGGAGGCTATGCGA- TAT-3 and5-TTCCCAAACACCACCTACGACA-3;IFNc,5-ATGTAT- GATGGGCAGTGTGG-3 and 5-CCAGGCGCAGTAACTGAAAT-3; 18S,5-TTGCCGCTAGAGGTGAAATT-3 and5-GCAAATGCTTTCGC- TTTCG-3; EF1␣, 5-TGCCCCTCCAGGATGTCTAC-3 and 5-CAC- GGCCCACAGGTACTG-3; VP2, 5-GCCAAGATGACCCAGTCCAT-3 and5-TGACAGCTTGACCCTGGTGAT-3.

2.8. Westernblotting

TOcellsweresplit2:3thedaybeforetransfection.Thecellswere transfectedat80%conﬂuenceusingthe10␮lNeon transfection

system(Lifetechnologies)withbufferRandatapulsevoltageof 1200,pulse width20ms, 2 pulses.450,000 cells wereusedfor each transfection.For each wellatotal of 350ng plasmidwere used.Aftertransfectionthecellswereseededin48-wellplatesin triplicateswithMEMgrowthmediumcontaining12%FBSwithout antibiotics,andincubatedwithCO2at20^◦C.Cellswereharvested 48hposttransfectionin40␮l2×SDSbuffer.15–20␮lofthesam- pleswereloadedineachwellofaprecast4–12%gradientNuPAGE NovexBis-TrisgelandsubjectedtoSDS–polyacrylamidegelelec- trophoresis(SDS–PAGE)with1×MOPS(Invitrogen)for45minat 200Vand120mA.ThemarkersMagicMark^TMXP(Invitrogen)and SeeBluePlus2Prestained(Invitrogen)weresimultaneouslyloaded formolecularweightestimations.Westernblottingofthesepa- rated proteinstoa polyvinylidene diﬂuoride(PVDF)membrane (Millipore)wasperformedusingtheInvitrogenNuPAGEsystem accordingtothemanufacturer’sinstructions.Ananti-Flagantibody (1:3000 dilution) (Sigma) for detection of Flag-tagged proteins wereusedandgoatanti-mouse-HRPantibody(1:5000)(SantaCruz Biotechnology)asasecondaryantibody.

2.9. Statisticalanalyses

Two-sidedunpairedStudentttestwasusedtocalculatestatis- tics, where p≤0.05 was considered to indicate a statistically signiﬁcantdifference.

3. Results

3.1. IPNVinfectiondoesnotinduceIFNatranscriptionincell culture

TostudytheeffectofIPNVinfectionontheIFNresponse,TO- cellswereinfectedwithIPNVatanMOIof10,andexpressionof IFN transcriptsand VP2transcriptsweremeasuredafter6–48h (Fig.1).ThecellsshowedbeginningCPEat48handstrongCPEat 72hafterinfection.TheIFNaexpressionshowedrelativelysmall changes in infected cells compared to uninfected control cells, whereonlyaminorincreaseinIFNatranscriptswasobservedat6h (1.5times)(Fig.1A).Asexpected,theexpressionoftheviralprotein VP2showedastrongincreasethroughoutthe48hinfectionperiod (Fig.1B).IPNVdoesthusnotinduceIFNaduringinfectionofTO cells,whichsupportsthatthevirusmayexpressIFNantagonizing proteins.

3.2. IPNVpreVP2,VP3,VP4andVP5proteinsinhibitIFNa1 promoteractivationwhileVP1activatestheIFNa1promoter

To measure the effect of IPNV proteins on IFNa1 promoter activation, we used a previously described reporter gene con- structwheretheAtlanticsalmonIFNa1minimalpromoterregion controls expression of a luciferase gene (Bergan et al., 2006).

As activator of the IFNa1 promoter, we used Atlantic salmon IPS-1, which is known to activate the IFNa1 promoter upon overexpression (Lauksund et al., 2009).IPS-1 is a key adapter protein in the RIG-I/MDA5 signaling pathway, which controls typeIIFNexpressioninmostcells.TO-cellswereco-transfected withthereporter plasmidand a plasmidcontainingoneof the IPNV genesand a plasmid expressing IPS-1.Luciferase activity in thecells wasmeasuredat48haftertransfection.Theresults showedthattheviralproteinspreVP2,VP3,VP4andVP5inhib- itedIPS-1mediatedactivationoftheIFNa1promoter(Fig.2A).The strongest inhibitoryeffectwasobservedwithVP4,which virtu- allyabolishedpromoter activation.In contrast,VP1surprisingly increasedactivationoftheIFNa1promoterwhenco-transfected with IPS-1. To ﬁnd out if VP1 could activate the IFNa1 promoter by itself, individual VP constructs were co-transfected

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Fig.1. ExpressionofIFNaandVP2inIPNVinfectedTOcellsmeasuredbyqPCR.QuantitativePCRshowingexpressionofIFNa(A)andVP2(B)inTOcellsinfectedwithIPNV (MOI10).GeneexpressionwasnormalizedagainstEF1␣.ExpressionwasdeterminedrelativetotranscriptlevelsinuninfectedTOcells(=1)forIFNa,andrelativetothe6h infectiontimepointforVP2(meanCt=31).Valuesaremean±SD(N=3).

with IFNa1 promoter construct into TO cells and assayed for luciferaseexpression.AsshowninFig.2B,VP1expressionalone didindeed activatetheIFNa1promoterwhile preVP2-VP5gave areductioninpromoteractivitycomparedwiththetransfection control.

Sinceactivation oftype IIFN transcriptionthrough theRIG- I/MDA5 pathway ultimately results in activation of IRFs, we next studied the effect of IPNV proteins on IRF1 and IRF3 mediated activation of the IFNa1 promoter. Overexpression of these IRFs haspreviously been shown to result in strongacti- vation of the IFNa1 promoter (Bergan et al., 2010). In these

experiments VP constructs were co-transfected with the promoter construct and an expression plasmid encoding either IRF1 or IRF3. The results showed that preVP2, VP3, and VP4 stronglyinhibitedbothIRF1andIRF3mediatedactivationofthe IFNa1 promoter. VP5 had inhibitory effect against IRF1 mediated activation of the promoter, but did not cause signiﬁcant inhibition of IRF3 mediated activation of the IFNa1 promoter (Fig.2CandD).

Taken together,thedatashowedthat preVP2, VP3,VP4 and VP5 wereallabletoinhibitIPS-1,IRF1andIRF3mediated acti- vationoftheIFNa1promoter.SinceweobservedIFNa1-promoter

Fig.2. EffectofIPNVproteinsonactivationoftheAtlanticsalmonIFNa1promoter.InallexperimentsTO-cellswereco-transfectedwithfourexpressionconstructs;a plasmidcontainingtheIFNa1promoterfusedtoaluciferasegene,aplasmidcontainingtheRenillaluciferasegeneasatransfectioncontrol,aplasmidcontaininganIPNV gene(VP1-VP5),andacontrolplasmidoraplasmidcontainingageneencodingasignalingproteinintheIFNa1inductionpathway.Sampleswereharvested48hafter transfectionandanalyzedforpromoteractivationbymeasuringluciferaseactivity.Resultsarepresentedasmeanrelativelightunits(RLU)±SD(N=3).(A)EffectofIPNV proteinsonIPS-1mediatedactivationofthepromoter.Significantdifferences(p<0.05)fromvectorcontrolandIPS-1indicatedby*and†,respectively.(B)Effectofthe individualIPNVproteinsonpromoteractivation.Significantdifference(p<0.05)fromvectorcontrolandIPS-1indicatedby*and†,respectively.(C)EffectofIPNVproteins onIRF1mediatedactivationofthepromoter.Significantdifferences(p<0.05)fromvectorcontrolandIRF-1indicatedby*and†,respectively.(D)EffectofIPNVproteinson IRF3mediatedactivationofthepromoter.Significantdifferences(p<0.05)fromvectorcontrolandIRF-3indicatedby*and†,respectively.(E)EffectofIPNVproteinsonVP1 mediatedactivationofthepromoter.Significantdifferences(p<0.05)fromvectorcontrolandVP1indicatedby*and†,respectively.

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Fig.3.Co-stimulatoryeffectsofVP1andIRFsonactivationoftheAtlanticsalmonIFNa1promoter.TO-cellswereco-transfectedwithaplasmidcontainingtheminimalIFNa1 promoterfusedtoaluciferasegenealongwithanexpressionconstructforRenillaluciferaseasatransfectioncontrol.Inaddition,expressionconstructsforIRF1orIRF3, VP1,andVP1incombinationwithIRF1orIRF3wereincluded.Resultsarepresentedasmeanrelativelightunits(RLU)±SD(N=3).(A)EffectsofIRF1,VP1,acombinationof VP1andIRF1andvectorcontrol.IRF1+VP1issigniﬁcantlydifferentfromVP1andIRF1(p<0.05).(B)EffectsofIRF3,VP1,acombinationofVP1andIRF3andavectorcontrol.

IRF3+VP1issigniﬁcantlydifferentfromVP1andIRF3(p<0.05).AllgroupsaredifferentfromvectorcontrolinAandB(p<0.05).

activatingpropertiesofVP1,wealsowantedtoexamineifVP2- VP5had inhibitoryeffectagainstthisactivation.Co-transfection ofTO-cellswithIFNa1promoterconstruct,VP1andeach ofthe otherIPNVgeneswasperformed.Theresultsshowedthatindeed, VP2–VP5werealsoabletoinhibitVP1mediatedactivationofthe IFNa1promoter(Fig.2E).

3.3. SynergisticeffectsofVP1andIRFsoninductionofIFNa1 VP1andIRF1orVP1andIRF3showedstrongsynergisticeffects in activationof theIFNa1 promoter. TransfectingTO cells with a combination of IRF1 and VP1 plasmids increased theactiva- tion capacity of VP1 12 times,while combining IRF3 withVP1 increasedtheactivationcapacityofVP1by10.5times(Fig.3Aand B).

SinceoverexpressionofVP1activatedtheIFNa1promoter,we wantedtostudywhetherVP1wasabletoinducetranscriptionof IFNeitheraloneorincombinationwithIRF1andIRF3.Accordingly, TO-cellsweretransfectedwithplasmidscontainingVP1aloneor VP1 togetherwithplasmidscontainingIRF1orIRF3.Expression ofIFNainresponsetoIPS-1plasmidwasusedasapositivecon- trolandpreVP2wasincludedasanegativecontrol.After24hthe cellswereharvested,andanalyzedforIFNa,IFNbandIFNctrans- cripts.TransfectionwithIPS-1resultedina6-foldup-regulation ofIFNatranscriptscomparedwiththecontrol(p<0.001)(Fig.4).

Up-regulationofIFNawashardlydetectableaftertransfectionwith VP1,IRF1orIRF3alonewhereasthecombinationofVP1witheither IRF1orIRF3showeda6–8timesup-regulationofIFNatranscript levels.ThepreVP2plasmidshowednosigniﬁcantup-regulationof IFNa1transcripts(p=0.8).NoneofthecellgroupsshowedIFNb transcriptsorup-regulationofIFNctranscripts(datanotshown).

3.4. AntiviraleffectinducedbyVP1andIRF1/IRF3isduetoIFNa SinceVP1togetherwithIRF1/IRF3activatedIFNatranscription, wewantedtotesttheirabilitytoinduceantiviralactivityinTO cells. The cells weretransfected withplasmids expressingVP1, IRF1,IRF3aloneorVP1incombinationwiththeIRFs,ortransfected withplasmidwithoutinsertforinfectedanduninfectedcontrols.

After 72h, the cell supernatants were harvested for antiviral assayof IFN whilethe cellswere infected withIPNVfor direct measurementofantiviralactivityonthetransfectedcells.When theinfected controlcellshadreachedfullCPE,thesupernatants

wereharvestedforviraltitrationandtheremainingcelllayerwas stainedwithcrystalvioletformeasurementofcellsurvival.The assay showedsigniﬁcant increasedcell survival for all plasmid transfections,butcellstransfectedwithcombinationofVP1and IRF1/IRF3plasmidsshowedhighestsurvival(Fig.5A).Thispattern ofantiviralactivitywasalsoobservedmeasuredasreductionin virustitersasshownbytheTCID50calculationsshownunderthe correspondingbarsofOD550measurements(Fig.5A).

WenextstudiedifIFNwassecretedbycellstransfectedwiththe differentplasmidcombinations.Forthispurposethesupernatant fromthetransfectedcellswasaddedtonewTOcellsinthepres- enceorabsenceofIFNa1antibody.Thecellswereincubatedfor 24hforinductionofantiviralproteinsandinfectedwithIPNV.Cell survivalwasmeasuredbycrystalvioletstainingwhentheinfected controlcellshadreachedfullCPE.AsshowninFig.5B,onlysuper- natantsfromcellstransfectedwithcombinationsofVP1andIRFs gavesigniﬁcantprotectionofcells.Theantiviralactivityofthese supernatantswascompletelyblockedbypre-treatmentwithIFNa1 antibody.

Fig.4.IFNaexpressioninTOcellsinresponsetooverexpressionofVP1incom- binationwithIRF1orIRF3.Cellsweretransfectedwithexpressionconstructsfor VP1,IRF1andIRF3incombinationwithplasmidcontrolorVP1incombinationwith IRF1orIRF3.CellstransfectedwithIPS-1orpreVP2wereusedaspositiveandneg- ativecontrol.RNAwasextractedafter24handIFNaexpressionwasdetermined byqPCRrelativetotranscriptlevelsinplasmidcontroltransfectedcells(=1).Gene expressionwasnormalizedagainst18SrRNA.Valuesaremean±SD(N=3).Aster- isksdenotesignificantdifferencesfromvectorcontrol(p<0.05).Cellstransfected withVP1+IRF1weresignificantlydifferentfromcellstransfectedwithIRF1+vector controlandfromVP1+vectorcontrol(p<0.005).CellstransfectedwithVP1+IRF3 weresignificantlydifferentfromcellstransfectedwithIRF3+vectorcontroland fromVP1+vectorcontrol(p<0.005).

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Fig.5.AntiviralactivityofVP1,IRF1andIRF3againstIPNV.(A)TO-cellsweretrans- fectedwithexpressionconstructsforVP1,IRF1,IRF3aloneorVP1incombination withIRF1orIRF3,oremptyvectorforcontrolsamples.Threedaysaftertransfection, supernatantsfromthetransfectedcellswereharvestedandkeptforuseintheIFN neutralizationassayshownin(B)whilethecellswereinfectedwithIPNV(MOI0.1).

WhenfullCPEwasobservedafter4days,thesupernatantswereharvestedforviral titration,andthesurvivingcelllayerwasstainedwithcrystalviolet.Cellsurvival wasdeterminedbymeasuringtheabsorbanceat550nm.Viraltitersinmedium supernatantsfromthedifferenttreatmentgroupsweredeterminedbytheTCID50

method.Allgroupsaredifferentfrominfectedcontrol(p<0.05).(B)Measurementof IFNactivityincellsupernatantsdescribedin(A)inthepresenceorabsenceofneu- tralizingantibodyagainstIFNa1.TO-cellsweretreatedwithsupernatantsfor24h andtheninfectedwithIPNVasdescribedfor(A).AtfullCPE,cellsurvivalwasdeter- minedbystainingcellswithcrystalvioletandmeasuringabsorbanceat550nm.

Asterisksdenotesigniﬁcantdifferencesfrominfectedvectorcontrol(p<0.05).

3.5. TheabilityofVP1toactivatetheIFNa1promoteris dependentonanintactserineinposition163

VP1istheIPNVpolymeraseandhasbeendemonstratedtopro- duceRNAfrombothviralandnon-viralsourcesinvitro(Graham etal.,2011).ItcanthusbehypothesizedthatVP1mightstimulate IFNsthroughsynthesisofdsRNA.Wewantedtotestthishypothesis bytransfectingTOcellswithamutantVP1thatwasdefectinRNA polymeraseactivity.ForthispurposewegeneratedaVP1sequence thatwasmutatedinposition163sincethismutant(VP1S163A)has beenshownneithertobeabletoguanylylateVP1nortobeableto producedsRNA(Petitetal.,2000).

VP1S163Awasco-transfectedintoTO-cellstogetherwiththe IFNa1-promoterconstructtoseeifthemutantVP1 wasableto activatethepromoter (Fig.6).Wild-type VP1wasincludedasa positivecontrol.TheresultsshowedthattheS163Amutationcom- pletelyabolishedVP1sabilitytoactivatetheIFNa1promoter.The

Fig.6.EffectoftheVP1mutantS163AonactivationoftheIFNa1promoter.TO- cellswereco-transfectedwithaplasmidcontainingtheminimalIFNa1promoter fusedtoaluciferasegenealongwithanexpressionconstructforRenillaluciferase asatransfectioncontrol.InadditionexpressionconstructsforVP1,themutant VP1S163A(withoutpolymeraseactivity),orvectorcontrolwereincluded.Results arepresentedasmeanrelativelightunits(RLU)±SD(N=3).Asterisksdenotegroups signiﬁcantlydifferentfromcellstransfectedwithvectorcontrol(p≤0.05).

VP1andVP1S163Aproteinswereexpressedatcomparablelevels whendetectedwithananti-ﬂagantibody(Fig.S2).

3.6. AntagonisticeffectofVP4isindependentofanintact proteolyticactivesite

TheobservationthatVP4wasabletoinhibitIPS-1,IRF1and IRF3 mediatedactivation of theIFNa1promoter, indicated that VP4actedontheIRFsoronsignalingmembersinvolved inIRF- phosphorylation.AnobviousquestionwasiftheIFNantagonistic effectofVP4wasduetoitsproteaseactivity(Duncanetal.,1987).

FirstwestudiedwhetherVP4mightdegradeIRF1andIRF3.How- ever,nodegradationoftheIRFscouldbedetectedinaWestern blotfollowingco-transfectionofVP4withtheIRF1orIRF3(data notshown).WethenwantedtotestifIFNantagonisticactivityof VP4wasdependentofitsproteaseactivitybycreatingaprotease- deadmutantofVP4.Sincemutationsinthesiteofthelysinegeneral baseK674havebeenshowntobeevenmoreefﬁcientinabolishing proteaseactivitythanmutatingthereactiveserineresidue(Petit etal.,2000),thissitewaschosenforasingleaminoacidmuta- tion.Thismutationhaspreviouslyshowntoabolishtheprotease activityofVP4withalltestedmutations(Petitetal.,2000).The expressedVP4andVP4K674Aproteinswerepresentincompara- bleamountswhendetectedwithananti-ﬂagantibody(Fig.S2).

TheconstructedVP4K674Awasfoundtobejustasefﬁcientasthe wildtypeVP4ininhibitionofIPS1-mediatedIFNa1promoteracti- vationinaco-transfectionassay(Fig.7).ThissuggeststhattheIFN antagonisticpropertyofVP4isindependentofitsproteaseactiv- ity.Wethenlookedforpotentialnon-covalentbindingofVP4to theIRFs.However,wedidnotobserveanybindingofIRF1/3to VP4inaco-immunoprecipitationassay(datanotshown).Asthere areanabundanceofproteinsandregulatorsinvolvedintheIRF branchofthesignalingcascade,therearemany otherpotential targetsforinhibition.Unfortunately,farfromallofthepotential signalingmembershavebeenclonedinAtlanticsalmon.

4. Discussion

IPNVreplicationisstronglyinhibitedinsalmoncellstreatedby typeIIFN(Robertsenetal.,2003;Svingerudetal.,2012).Thevirus mustthereforebedependentoninhibitionofIFNinductionduring infection.ThisissupportedbytheobservationthatIPNVinfectionof TOcellsonlyresultedinverysmallchangesintheamountsofIFNa

(7)

Fig.7.EffectoftheVP4mutantK674AonactivationoftheIFNa1promoter.TO-cells wereco-transfectedwithaplasmidcontainingtheminimalIFNa1promoterfusedto aluciferasegenealongwithanexpressionconstructforRenillaluciferaseasatrans- fectioncontrol.InadditionexpressionconstructsforVP4,themutantVP4K674A (withoutproteaseactivity),oravectorcontrolwereincluded.Resultsarepresented asmeanrelativelightunits(RLU)±SD(N=3).Asterisksdenotegroupssigniﬁcantly differentfromcellstransfectedwithvectorcontrol(p≤0.05).

transcripts(Fig.1).Moreover,thepresentworkdemonstratesthat IPNVencodesproteinswithpowerfulIFNantagonisticproperties wherealloftheproteinsencodedbyIPNVsegmentBwereableto inhibitinductionofIFNa1throughtheRIG-I/MDA5signalingpath- way.OverexpressionofpreVP2,VP3,VP4andVP5allinhibitedIPS-1 mediatedactivationof theIFNa1promoter. WhileVP4 virtually abolishedIFNa1promoteractivation,preVP2,VP3andVP5alldis- playedastrongalbeitnotcompleteinhibition.Theseviralproteins arelikelytoactonsignalingfactorsdownstreamofIPS-1sincethey allinhibitedactivationoftheIFNa1promotermediatedbyoverex- pressionofIRF1andIRF3.Themechanismsofinhibitionobserved bypreVP2,VP3andVP5remaintobeelucidated.ThedsRNAbind- ingabilityofVP3haspreviouslybeenproposedasamechanismfor avoidingantiviralhostresponses(Pedersenetal.,2007).Binding dsRNAis,however,notlikelytobethemainmechanismofantag- onismobservedinthepresentstudies,astheassaysarebasedon overexpressionofproteinsfromDNAvectors,andtheproteinlev- elsofthetransfectioncontrol,Renillaluciferase,werenotaffected bythepresenceofVP3.ItisthuspossiblethatVP3hasmultiple IFNantagonisticproperties,awell-knowntraitinviruses.Oneof themoststudiedviralproteinswithmultiplefunctionsasanIFN antagonististhenon-structuralNS1proteinofinﬂuenzaAviruses.

InadditiontobeingimportantforenhancingviralmRNAtransla- tion,NS1isabletoablatethehostinnateimmunesystembothby limitingtheinductionofIFNbyblockingRIG-Iactivation,andby directlyinhibitingantiviralproteinssuchasPKRandOAS/RNaseL.

NS1alsoinhibitsexportofmRNAfromthenucleus(reviewedin Haleetal.,2008).

SinceVP4possessesproteaseactivity,itwassuspectedthatit mightcleavemembersoftheRIG-I/MDA5-signalingpathwaysim- ilartoseveralotherviralproteases.Forinstancethepicornavirus 3CprocleavesRIG-I(Barraletal.,2009),andtheNS3/4Aserinepro- teaseofhepatitisCvirus,theNS3/4AproteaseofGBvirusBand the3ABCofhepatitisAvirusallcleavesIPS-1(Chenetal.,2007;

Lietal.,2005;Yangetal.,2007).However,VP4mutatedtoabol- ishproteaseactivity(K674A),retaineditsabilitytoinhibitIPS-1 mediatedactivationoftheIFNa1promoter.Neithercouldwedetect anydegradationproductsofIRF1orIRF3whenco-transfectedinto cellswithVP4.TheproteaseactivityofVP4isthusnotlikelytobe importantforitsIFNaantagonisticactivityintheRIG-I/MDA5path- waydownstreamofIPS-1.AsinteractionsbetweenVP4andIRF1 orIRF3werenotobserved,VP4islikelytoactonothersignaling membersinvolvedinactivationoftheIRFs.PotentiallyVP4might

preventphosphorylationof theIRFs. Otherpossibilitiesarethat VP4mightmodifythefunctionoftheIRFsbypromotingorrevers- ingubiquitination,SUMOylationorISGylationonthemembersof thesignalingcascade,similarmodiﬁcationshavebeenshownof other antagonisticviralproteinssuchas inﬂuenzaA virusNS1, murinehepatitisvirusNSP3andebolavirusVP35(Gacketal.,2009;

Kubotaetal.,2009;Zhengetal.,2008).Ithasbeenshownthatthe IFNantagonisticactivityofthepapain-likeprotease(PLP)ofthe humancoronavirusNL63isindependentofitsenzymaticactivity (Clementzetal.,2010).ThePLPisbothaproteaseandadeubiq- uitinase,abletoprocessbothK-48andK-63linkedpolyubiquitin chains.Ablatingthesefunctionseitherbymutationorbyaddinga proteaseinhibitordidnotinhibittheantagonisticnatureofthepro- tein.AlsotheproteaseofthemurinehepatitisvirusactsasanIFN antagonist,andeveniftheantagonisticactivityispartlydependent onthedeubiquitinaseactivity,theproteasedefectivemutantsstill retainedsomeantagonisticactivity(Zhengetal.,2008).Likethese viralproteases,theIFNantagonisticeffectofIPNVVP4seemstobe independentofanintactcatalyticactivity.

Thebirnavirusinfectiousbursaldiseasevirus(IBDV)isstruc- turally related to IPNV, but causes acute immunosuppressive diseaseinbirdsduetoinfectionofBcells(Mahgoubetal.,2012).

Even if the immunosuppressive nature of IBDV has been well established,themolecularbasisfor thismechanism isnot fully understood.ItwasrecentlyreportedthattheVP4proteaseofIBDV isinhibitingIFNexpressionbyinteractingwiththeglucocorticoid- inducedleucinezipper(GILZ)(Lietal.,2013).GILZhaspreviously beenshowntobindbothNF-␬BandIRF3,inhibitinggenetranscrip- tiondirected bythesetranscription factors(Ogawaet al.,2005;

Reilyetal.,2006).WhetherIPNVVP4utilizesasimilarmechanism astheIBDVVP4inIFNsuppression,isasubjectforfuturestudies.

AsurprisingfindinginthepresentworkwastheIFNinducible propertiesoftheviralpolymeraseVP1.OverexpressionofVP1alone potently activatedtheIFNa1promoter and increasedactivation mediatedbyoverexpressionofIRF1andIRF3.Moreover,overex- pressionofVP1incombinationwithIRF1orIRF3up-regulatedIFNa transcription andincreasedantiviralactivityagainstIPNV inTO cells.TheantiviralactivityfromthetransfectedcellswithVP1in combinationwithIRF1orIRF3wasduetoIFNasincetheantiviral activityofthesupernatantscouldbecompletelynegatedbythe additionofIFNa1antibody.TheIFNainducingpropertiesofVP1 maybeduetosynthesisofRNAsinceithasbeendemonstratedthat VP1producesRNAfrombothviralandnon-viralsourcesinvitro (Grahametal.,2011).Thishypothesisissupportedbythefactthat onesingleaminoacidsubstitutioninposition163abolishedthe abilityofVP1toactivateIFNa1promoteractivation.TheVP1S163A mutanthasbeenshownneithertobeabletoguanylylatenortopro- ducedsRNA(Xuetal.,2004).Eventhoughthe163siteisnotthe actualguanylylationsite(Grahametal.,2011),thismutantisdefi- cientinproducingdsRNA(Xuetal.,2004).RNAsynthesizedbyVP1 mayberecognizedbyRIG-IorMDA5andthustriggeractivationof theIFNa1promoter.DuringIPNVinfectionbothviraldsRNAand non-specificRNAspeciesformedbyVP1maythuspotentiallybe recognizedbyRIG-I/MDA5riskingactivationofIFNtranscription.

IPNVmaythushavedevelopedmultipleIFNantagonizingmecha- nismstoavoidtriggeringofIFNsynthesisbyVP1sincethisvirusis highlysensitivetotheantiviralactivitiesinducedbytypeIIFNsin cells.dsRNAcouldnotbedetectedbyadsRNAantibodyaftertrans- fectingcellswithVP1(datanotshown),butantibodybindingmight bedependentofsizesofdsRNAmoleculesthatarelargerthanthe onesproducedbyVP1.TheconcentrationofsuchRNAmolecules isalsolikelytobelow,sinceVP1ishighlydependentonanIPNV speciﬁcprimermoleculetostartsynthesisofdsRNA(Dobos,1995).

Inconclusion,Atlanticsalmoncellsseemtohavetheabilityto establishanantiviralstatebyrecognizingdsRNAproducedbythe IPNVVP1.Toovercomethispotentialbarrierofinfection,IPNVhas