La Piedad Michoacán Mexico Virus V protein antagonizes type I interferon response by binding STAT2 protein and preventing STATs nuclear translocation

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VirusResearch213(2016)11–22

ContentslistsavailableatScienceDirect

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

La

Piedad

Michoacán

Mexico

Virus

V

protein

antagonizes

type

I

interferon

response

by

binding

STAT2

protein

and

preventing

STATs

nuclear

translocation

Giuseppe

Pisanelli

a,b,d

_,

_Maudry

_{Laurent-Rolle}

a,b

_,

_Balaji

_Manicassamy

a,b,1

_,

Alan

Belicha-Villanueva

a,b

_,

_Juliet

_Morrison

a,b,2

_,

_Bernardo

_{Lozano-Dubernard}

e

_,

Felipa

Castro-Peralta

e

_,

_Giuseppe

_Iovane

d

_,

_Adolfo

_{García-Sastre.}

a,b,c,∗

a_Department_of_{Microbiology,}_Icahn_School_of_Medicine_at_Mount_Sinai,_One_Gustave_L._Levy_Place,_New_York,_NY_10029,_United_States

b_Global_Health_and_Emerging_Pathogens_Institute,_Icahn_School_of_Medicine_at_Mount_Sinai,_One_Gustave_L._Levy_Place,_New_York,_NY_10029,_United_States c_Department_of_Medicine,_Division_of_Infectious_Disease,_Icahn_School_of_Medicine_at_Mount_Sinai,_One_Gustave_L._Levy_Place,_New_York,_NY_10029,_United States

d_Department_of_Veterinary_Medicine_and_Animal_Production,_University_of_Naples_Federico_II,_Via_Federico_Delpino_1,₈₀₁₃₇_Naples,_Italy e_Departamento_de_{Investigación}_y_Desarrollo,_Laboratorio_Avi-Mex,_SA_de_CV,_Bartolache_1862,_Colonia_del_Valle,_D.F._México_01900,_Mexico

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received28June2015

Receivedinrevisedform30October2015 Accepted30October2015

Availableonline3November2015 Keywords:

(LPMV)LaPiedadMichoacanMexicoVirus LPMV-V

STAT2

Interferon-signalingantagonist

a

b

s

t

r

a

c

t

LaPiedadMichoacánMexicoVirus(LPMV)isamemberoftheRubulavirusgenuswithinthe Paramyx-oviridaefamily.LPMVistheetiologicagentof“blueeyedisease”,causingasigniﬁcantdiseaseburden inswineinMexicowithlong-termimplicationsfortheagriculturalindustry.Thisvirusmainlyaffects pigletsandischaracterizedbymeningoencephalitisandrespiratorydistress.Italsoaffectsadultpigs, causingreducedfertilityandabortionsinfemales,andorchitisandepididymitisinmales.

VirusesoftheParamyxoviridaefamilyevadetheinnateimmuneresponsebytargetingcomponents oftheinterferon(IFN)signalingpathway.TheVprotein,expressedbymostparamyxoviruses,isa well-characterizedIFNsignalingantagonist.Untilnow,therewerenoreportsontheroleoftheLPMV-Vprotein ininhibitingtheIFNresponse.InthisstudywedemonstratethatLPMV-VproteinantagonizestypeIbut nottypeIIIFNsignalingbybindingSTAT2,acomponentofthetypeIIFNcascade.Ourresultsindicate thatthelast18aminoacidsofLPMV-VproteinarerequiredforbindingtoSTAT2inhumanandswine cells.WhileLPMV-VproteindoesnotaffecttheproteinlevelsofSTAT1orSTAT2,itdoespreventthe IFN-inducedphosphorylationandnucleartranslocationofSTAT1andSTAT2therebyinhibitingcellular responsestoIFN␣/␤.

1. Introduction

La PiedadMichoacán Mexico Virus (LPMV) is a member of theRubulavirusgenuswithintheParamyxoviridaefamily,inthe orderofMononegavirales.Thevirus(LPMV)wasﬁrstdiscovered inswine duringtheearly1980s incentral-west Mexico,where

∗ Correspondingauthorat:DepartmentofMicrobiology,IcahnSchoolofMedicine atMountSinai,OneGustaveL.LevyPlace,NewYork,NY10029,UnitedStates. Fax:+12125341684.

E-mailaddress:adolfo.garcia-sastre@mssm.edu(A.García-Sastre.).

1 _Present_address:_Department_of_{Microbiology,}_University_of_Chicago,₉₂₀_East 58thStreet,CLSC711B,Chicago,IL60637,UnitedStates.

2 _Present_address:_Department_of_{Microbiology,}_School_of_Medicine,_University_of Washington,RosenBuilding,960RepublicanSt.,Seattle,WA98109-4325,United States.

themajorityofthenation’spigfarmsarelocated(Escobar-Lopez et al., 2012; Moreno-Lopez et al., 1986; Stephan et al., 1988). Thisswinevirusisthecausativeagentofblueeyedisease(BED) thatappearswithavariedsymptomatologydependingontheage of theanimals. Thepiglets exhibit neurologicaland pulmonary symptomscharacterizedbyencephalitis, cornealopacity and/or pneumonia(Mendoza-Maganaetal.,2007;Rivera-Benitezetal., 2013;Rodriguez-Roponetal.,2003;Wimanetal.,1998).Inadults, thediseaseischaracterizedbysymptomsthataffectthe repro-ductivesystem.Sowsexhibitincreasedoestrus,andanincreased incidenceofstillbirthandmummiﬁedfetuses (Hernandezetal., 1998;Hernandez-Jaureguietal.,2004).Inboars,the symptomatol-ogyischaracterizedbylowspermquality,orchitisandepididymitis thatcanleadtopermanentsterility(Cuevas-Romeroetal.,2014; Ramirez-Mendozaetal.,1997;Solisetal.,2007).BED isamong

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thetopfourdiseasesthatafﬂicttheMexicanswineindustry.The reductionofreproductiveperformanceandthemortality associ-atedwithLMPVinfectionsinpigscausesigniﬁcanteconomiclosses (Escobar-Lopezetal.,2012).

LPMVis anenvelopedviruswitha single-stranded negative-senseRNAgenomeofapproximately15Kb.Thegenomecontains6 genesencodingatleast9proteins:thenucleoprotein(NP),matrix (M),fusion(F),hemagglutinin-neuraminidase(HN),largeprotein (L)andphosphoprotein(P)(Bergetal.,1997,1991;Cuevas-Romero etal.,2013;Linneetal.,1992;Reyes-Leyvaetal.,1999; Sanchez-Betancourtetal.,2012;Sundqvistetal.,1990;Svendaetal.,1997; Zenteno-Cuevasetal.,2007).ThePgeneoftheparamyxoviruses encodeseveralproteinsbyusingdifferentinitiationcodonsand by a unique mechanism involving RNA editing that generates alternatemRNAs by site-specific co-transcriptionalinsertion of non-templatednucleotides(Hausmannetal.,1999a,b;Isenietal., 2002;Kolakofskyetal.,2005;Thomasetal.,1988).Inparticularthe PgeneofLPMVpossessesseveralopenreadingframes(ORFs).The useofalternativeinitiationcodonsfromthePgenemRNAresults inthegenerationofasmall126longaminoacidproteincalledC aswellasofP/V/WproteinsLPMVVproteinis249aminoacids inlengthanditsfirst168aminoacidsareidenticaltothoseofP andWproteins.Co-transcriptionaleditingatposition correspond-ingtoaminoacid168ofVresultsinnontemplateadditionofone ortwoGresiduesandinmRNAsencodingW(174a.a.)andP(404) proteinsrespectively.TheC-terminalregionoftheLPMVVprotein (LPMV-V)is60%similartomumpsvirusVprotein,56%similarto parainfluenzavirustype5(PIV5)Vprotein(PIV5-V),51%similar tohumanparainfluenzavirustype4Vprotein(hPIV4–V)and49% similartohumanparainfluenzavirustype2Vprotein(hPIV2-V)at theaminoacidlevel(Bergetal.,1992;Sundqvistetal.,1992).

The interferon (IFN) system plays a key role in the innate immunedefenseagainstviralinfections.Inmostcelltypes,the pro-ductionoftypeIIFN(IFN-␣/␤)isinitiatedimmediatelyafterviral infectionleadingtothesubsequentactivationoftheIFN-_␣/␤ signal-ingcascadetypeIIFNsignalingisactivatedbythebindingofIFNto itsreceptors(IFNAR1andIFNAR2)resultinginheterodimerization oftheIFNARreceptorsandtheresultingactivationofthe associ-atedJanuskinases,Tyk2andJak1(Colamonicietal.,1994,1995) (13).The activatedJanuskinases phosphorylateSTAT2(Nadeau etal.,1999;Yanet al.,1996)ontyrosine690(Greenlundetal., 1995)andSTAT1(Guptaetal.,1996;Qureshietal.,1995)on tyro-sine 701(Shuaiet al., 1993). PhosphorylatedSTAT1and STAT2 heterodimerizeandassociatewithinterferonregulatoryfactor9 (IRF9)toformIFN-stimulatedgenefactor3(ISGF3)(Fuetal.,1990), which translocateto thenucleus and binds the IFN-stimulated responseelements(ISREs)withininterferon-stimulatedgene(ISG) promoterstherebyinducingtheexpressionofmorethan100ISGs toestablishanantiviralstatethatlimitsviralreplicationand dis-semination (Childs et al., 2012; Garcia-Sastre and Biron, 2006; Garcia-Sastreetal.,1998;Laurent-Rolleetal.,2014;Manicassamy etal.,2010; Morrisonetal.,2013; Motzetal.,2013; Rajsbaum etal.,2014;RandallandGoodbourn,2008).TypeIIIFN(IFN-␥)is producedbyactivatedimmunecells (BilliauandMatthys,2009; Meyer,2009)andleadstotheproductionofadifferentsubsetof ISGsviaadistinctsignalingpathway.TypeIIIFN(IFN␥)bindsto dif-ferentreceptorcomplexalsoformedoftwosubunitsIFN-␥R1,and IFN-␥R2.TypeIIIFNsignalingactivatesSTAT1,byphosphorylation, whichhomodimerizestoformtheinterferongammafactor(GAF), whichtranslocatestothenucleusandbindstoDNAat␥-activated sequence(GAS)elements.

MostParamyxovirusesanalyzedsofarlikeNipahvirus,Hendra virus(Rodriguezetal.,2002,2003),PIV5(Didcocketal.,1999a,b; Preciousetal.,2005a),hPIV2(Parisienetal.,2001),mumpsvirus (Kubotaetal.,2005),caninedistempervirus(CDV)(Rothlisberger etal.,2010)withtheonlyexceptionofhPIV4(Nishioetal.,2005b),

areabletosubvertbothtypeIandtypeIIIFN-mediatedantiviral responsesbytargetingtheirsignalingcascades.

SinceParamyxovirusIFNantagonisticactivityismainlylinked totheVproteins,weexaminedwhethertheVproteinofLPMV alsohadtheabilitytoantagonizeIFNsignaling.Inthisstudy,we showthatLPMV-VproteinevadestypeIIFNsignalingbybinding STAT2andpreventingphosphorylationofSTAT2andSTAT1.Asa result,theSTATproteinsareretainedinthecytoplasm,preventing IFN-inducedSTATsactivationandnucleartranslocation,thereby inhibitingcellularresponsestoIFN␣/␤.Wealsoshowthatthelast 18aminoacidsofLPMV-VarerequiredforinhibitingIFNsignaling.

2. Materialandmethods

2.1. Cells

293T,HeLa,PK13andPK15cellsweregrowninDulbecco’s mod-iﬁedEaglemedium(Invitrogen)supplementedwith10%fetalcalf serum(FCS),penicillin,andstreptomycin. Allcells werekeptat 37◦Cinthepresenceof5%CO2.

2.2. Plasmids

LaPiedadMichoacánMexicoVirusVopenreadingframe(ORF), wasampliﬁedbyreversetranscriptionPCRofRNAextractedfrom LPMV-infectedcellswithMGG55strain.Primersareavailableupon request. PCRamplicons were cloned intopCAGGS (Niwaet al., 1991)and fromhere onare referred toas pCAGGS-LPMV-V.A HA-taggedversionofthesameampliconwasconstructedby plac-ingahemagglutinin(HA)tagsequenceencodingtheaminoacids MYPYDVPDYAdownstreamoftheVprotein,fromhereonreferred toaspCAGGS-LPMV-V-HA.Inordertodeterminetheregionsof interactionbetweenLPMV-VandSTATproteins, fourHA-tagged expressionvectorsweregeneratedlackingtheC-terminal18,25,50 and75aminoacidsandfromhereonarereferredtoasV-C18-HA, V-C25-HA,V-C50-HA,V-C75-HArespectively.

2.3. Reporterassays

293Tcells(1.5×106₎_were_transfected _in _6-well_plates _with

0.5␮gofaconstructhavinganISRE54promoterdrivingthe expres-sionofa fireflyreportergene (pISRE54-firefly-luciferase),0.1␮g ofaconstitutivelyexpressingrenillaluciferasereporterconstruct (pCAGGS-ren-luc),and3␮goftheexpressionplasmids. Twenty-fourhourspost-transfection,cellswerewashedandtreatedwith 1000U/mlIFNbeta1a(PBL11410-2).Sixteenhourspost-IFN treat-ment,cellswereharvestedusingreporterlysisbuffer(Promega,cat noE2810)andanalyzedforluciferaseactivitiesnormalizedwith renillaactivity.ForIFN␥-dependentgene expression,a reporter having3copies ofthe␥activatedsequence drivingthe expres-sionoffireflyluciferase(GAS-Luc)(0.5␮g)wastransfected with 0.1␮g of a constitutively expressing renilla-luciferase reporter construct (pCAGGS-ren-luc), and the indicated amounts of the expression plasmids. Twenty-four hourspost-transfection, cells were washed and treated with IFN-␥ (5ng/ml) (PBL). Sixteen hourspost-IFN treatment, cellswere harvested thenlysed, and theluciferaseactivitywasmeasuredbyapplyingadual-luciferase reporter(DLR)assaysystem(Promega)accordingtothe manufac-turer’srecommendation. Theluminescencesignalsofthefirefly andtherenillaluciferaseweremeasuredwithaTD-20/20 Lumi-nometer(Promega),andtheirratiowascalledrelativeluciferase activity,withtheratioover theempty vectorpCAGGS setto1 andanalyzedusingaDLRassay(Promega).Theassayswere per-formedintriplicateandp-valueswerecalculatedbyatwo-tailed Student’st-testforunpairedsamplesusingthesoftwareGraphPad Prism(GraphPadSoftware,Inc.).

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G.Pisanellietal./VirusResearch213(2016)11–22 13

Fig.1.LPMV-Vproteinexpressionandcellularlocalization.(A)293TcellsweretransfectedwithexpressionvectorsforHA-taggedPIV5-V,NipahV-VorLPMV-V,andlysates wereseparatedbySDS-PAGE(12%polyacrylamide).VproteinswerevisualizedbyimmunoblottingwithantibodiestotheHAepitopetag.Westernblotanalysisshows LPMV-V-HAexpressionprotein,withtheexpectedmolecularmassabout42kDa.(B)HeLacellsweretransfectedwithexpressionvectorsforHA-taggedNipahV-VorLPMV-V. Cellswereﬁxed,permeabilized,andstainedwithantibodiestoHA,asdescribedinSection2(materialsandmethods).NuclearDNAwasstainedwithDAPI.Imageswere obtainedusingconfocalmicroscopy.

Alternatively 293T cells were co-transfected with 3␮g of LPMV-V-HA,Nipah-V-HAandPIV5-V-HAplasmidsencodingthe respectivelyviralVproteins,250ngoftheIFN-inducible chloram-phenicolacetyltransferase(CAT)reporter(ISG54-CAT)and 50ng ofa plasmidconstitutivelyexpressingthefireflyluciferase pro-tein.24hposttransfectioncellsweretreatedwith1000U/mlof humanIFN-␤(PBL).24hposttreatment;cellswerelysedandCAT andLuciferaseactivityweremeasured.APhosphoimagerwasused toquantifyCATactivityandthevaluewasnormalizedtofirefly luciferaseactivity.Thefoldinductionofeachsamplewasthen cal-culatedastheCATactivityoftheIFN-treatedsamplenormalized tothefireflyluciferasevalueofthatsample.Thatvaluewasthen dividedbythenormalizedvalueof theuntreatedemptyvector transfectedcells.

2.4. NDV-GFPbioassay

PK13cellsweretransfectedwitheithertheemptypCAGGS plas-midor plasmids encoding various viral proteins as detailed in speciﬁcexperiments.Empty pCAGGSwasusedasnegative con-trolforIFNantagonism,wherePIV5-V-HAproteinwasincludedas positivecontrol.At24hpost-transfection,cellsweretreatedwith

1000U/mlofhumanIFN␤(PBL).Following24hofIFN␤ treat-ment,cellswereinfectedwithNDV-GFPasdescribedpreviously (Parketal.,2003).Fluorescenceimageswereobtainedat14h post-infection.In parallel experiments,VEROcells were ﬁrsttreated with1000U/mlofhumanIFN␤(PBL)for20h,thenthecellswere transfectedwitheithertheemptypCAGGSplasmidorLPMV-V-HA plasmid.24hposttransfectionthecellswereinfectedwith NDV-GFPdescribedpreviously(Parketal.,2003).Fluorescenceimages weretakenat14hpost-infection.

2.5. Real-timePCR

293Tcellswheretransfectedwithourexperimentaland con-trolplasmidsand24hlaterthecellswherewashedandtreated with1000U/mlIFN ␤orwithIFN-␥(5ng/ml) foranadditional 12h.TotalcellularRNAwasthenextractedusingTrizol(Invitrogen) andcDNAwasgeneratedbyreversetranscriptionusingrandom hexamersandoligodTprimerswithSuperscriptIIIreverse tran-scriptase(Invitrogen).Real-timePCRswerecarriedoutusingSYBR greensupermix(BioRad).MxA,ISG15,IP10andIRF1relativegene

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Fig.2.LPMV-VproteininhibitstypeIIFNsignalingbutnottypeIIIFNsignaling.(A)293TCellswereco-transfectedwithpCAGGSplasmidsencodingNipahV-V-HA, PIV5-V-HAandLPMV-V-HAproteins,plusanemptyvector(pCAGGS),aplasmidencodinganISRE-54-CATreporterandwithaplasmidconstitutivelyexpressingfirefly-luciferase usedascontrol.At24hposttransfection,cellsweretreatedwithIFN-␤(1000U/ml)for24hpriortoassayingforCATactivity.InductionofCATactivitywasnormalized tofireflyluciferaseactivity.(B)293TCellswereco-transfectedwithpCAGGSplasmidsencodingNipahV-V,PIV5-VandLPMV-Vproteins,plusanemptyvector(pCAGGS), GAS-luciferaseplasmidandaplasmiddrivingtheconstitutiveexpressionofrenilla-luciferaseusedasinternalcontrol.24hposttransfectionthecellsweretreatedwithIFN-␥ (5ng/ml)(PBL)and24hlatercellswerelysedandanalyzedforGAS-luciferaseactivitynormalizedtorenillaluciferaseactivity.(C)PK13cellsweretransfectedwitheither theemptypCAGGSplasmidorplasmidsencodingPIV5-V-HAandLPMV-V-HAviralproteins.EmptypCAGGSwasusedasnegativecontrolforIFNantagonism,whereasthe PIV5-V-HAproteinwasincludedaspositivecontrol.At24hpost-transfection,cellsweretreatedwith1000U/mlofhumanIFN␤(PBL).Following24hofIFN␤treatment, cellswereinfectedwithNDV-GFPasdescribedpreviously(Parketal.,2003).Fluorescenceimageswereobtainedat14hpost-infection.(D).VEROcellswerefirsttreated with1000U/mlofhumanIFN␤(PBL)for20h,thanweretransfectedwitheithertheemptypCAGGSplasmidorLPMV-V-HAplasmid.24hposttransfectionthecellswere infectedwithNDV-GFPasdescribedpreviously(Parketal.,2003).Fluorescenceimagesweretakenat14hpost-infection.

expressionwascalculatedusingthecomparativethresholdcycle method,employing18SrRNAandGAPDHasreferences.

2.6. Westernblot

Forpreparationofcellextracts,293TandPK15cellswere cul-turedinsix-wellplatesandtransfectedwithourexperimentaland controlplasmids.At48hpost-transfection,thecellsweretreated (ormock treated)with1000U/mlIFN- ␤orIFN-␥(5ng/ml) for 30minat 37◦C and then washedwith coldPBS and incubated oniceandsubsequentlylysedwith250_␮lofwhole-cellextract buffer(50mMTris[pH8.0],280mMNaCl,0.5%IGEPAL,0.2mM EDTA,2mMEGTA,10%glycerol,1mMdithiothreitol[DTT])

sup-plementedwithproteaseinhibitorcocktail(Complete;Boehringer Mannheim)and sodium vanadate (0.1mM). Lysateswere incu-batedonicefor10minandcentrifugedfor10minat20,000×g at 4◦C. Supernatantswere analyzed onsodiumdodecyl sulfate (SDS)4–15%polyacrylamidegels.Proteinswerethentransferredto nitrocellulosemembranesandwereprobedwithantibodiesagainst STAT1(UpstateBiotechnology,no.06-501),STAT2(SantaCruz,no. sc-476),phosphotyrosine701-STAT1(CellSignalingTechnologies, no.7649),Anti-phospho-STAT2(Tyr689)(Milliporeno.07-224), anti-actin(Sigma–Aldrich#A5060) anti␤tubulin(Cell Signal-ing#2146)andanti-HAepitopetag(Sigma–Aldrich#H3663),and visualizedbychemiluminescence(NENLifeSciences).

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Fig.3.LPMV-VproteindampenstheinductionoftypeIinterferon-stimulatedgenesISG15andMXAbutdoesnotreducetheinductionoftypeIIinterferon-stimulatedgenes IP10,IRF1.(AandB)293Tcellsweretransfectedwithourexperimentalandcontrolplasmidsand24hlaterthecellswerewashedandtreatedwithIFN␤at1000U/mlforan additional12h.TotalcellularRNAwasthenextractedandcDNAwasgeneratedbyreversetranscription.Real-timePCRswerecarriedoutusingSYBRgreensupermix.MxA handISG15relativegeneexpressionwascalculatedusingthecomparativethresholdcyclemethod,employing18SrRNAandGAPDHasReferences.(CandD)293Tcellswere transfectedwithourexperimentalandcontrolplasmidsand24hlaterthecellswerewashedandtreatedwithIFN-␥(5ng/ml)for12h.TotalcellularRNAwasthenextracted andcDNAwasgeneratedbyreversetranscription.Real-timePCRswerecarriedoutusingSYBRgreensupermix.IP10andIRF1relativegeneexpressionwascalculatedusing thecomparativethresholdcyclemethod,employing18SrRNAandGAPDHasreferences.

2.7. Co-immunoprecipitation

293Tcellswereseededin6-wellplates,andthenextdaycells weretransfected withLPMV-V-HA,Nipah-V-HAand PIV5-V-HA plasmids.At48hpostransfection,thecellsweretreated(orleft untreated)with1000U/mlIFN- ␤for 30minat 37◦C and then washedwithice-coldphosphate-bufferedsaline(PBS)and subse-quentlylysedwith250␮lofwhole-cellextractbuffer(50mMTris [pH8.0],280mMNaCl,0.5%IGEPAL,0.2mMEDTA,2mMEGTA,10% glycerol,1mMdithiothreitol[DTT])supplementedwithprotease inhibitorcocktail(Complete;BoehringerMannheim)andsodium vanadate(0.1mM).Aftera20-mincentrifugationat20,000×gat 4◦C, aliquotsfortotalprotein analyseswereseparated,andthe remainingsupernatantswereincubatedfor3hwiththeRed Anti-HAafﬁnitygelantibody(Sigma–AldrichCat#E6779)orwithout antibody.Alternativelythesupernatants wereincubatedfor3h withtheAnti-STAT2Antibodyfollowedbytheadditionofprotein

G-Sepharosebeadsat4◦Covernight.Aftera1-mincentrifugation at5000_×gat4◦C,thepelletswerewashedsixtimeswith whole-cellextractbufferandﬁnallydissolveddirectlyinLaemmlisample bufferforwesternblottingperformedasdescribedabove

2.8. Indirectimmunoﬂuorescence

Forindirectimmunofluorescenceexperiments,HeLacellswere grown to 70–80% confluence, oncover slides in 6-well plates, and 24h later were transfected with empty vector, LPMV-V-HA or NipahV-V-HA expression plasmids using lipofectamine 2000(Invitrogen)followingthemanufacturer’srecommendations. Beforefixation,cells weretreated for30minwith1000U/mlof IFN␤.At48haftertransfection,thecellswere,washedwithPBS andfixedwith500_␮lofice-coldmethanol-acetonesolution(1:1, V/V)for15min.FollowingPBSwashescellswerepermeabilyzed with0.5%IGEPALfor10minatroomtemperature.After3washes

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Fig.4. LPMV-VproteininhibitstypeIIFNinducedphosphorylationofSTAT1andSTAT2butnottypeIIIFN-dependentphosphorylationofSTAT1andpreventsSTAT1and STAT2nucleartranslocation.(A)293Tcellsweretransfectedwithemptyvector,LPMV-V-HA,NipahV-V-HAorPIV5-V-HAexpressionplasmids.Cellsweretreatedfor30min with1000U/mlofIFN␤priortolysis.LysateswereseparatedbySDS-PAGE(12%polyacrylamide),transferredtonitrocellulose,andvisualizedbyimmunoblottingwith antiseratoSTAT1,STAT2,phosphotyrosine701-STAT1,phosphotyrosine690-STAT2,anti-actinandanti-HAepitopetagasdescribeinSection2(materialsandmethods).(B) 293Tcellsweretransfectedwithemptyvector,LPMV-V-HA,orPIV5-V-HAexpressionplasmids.CellsweretreatedwithIFN-␥(5ng/ml)per30minpriortolysis.Lysates wereseparatedbySDS-PAGE(12%polyacrylamide),transferredtonitrocellulose,andvisualizedbyimmunoblottingwithantiseratoSTAT1,STAT2,phosphotyrosine 701-STAT1,phosphotyrosine690-STAT2,anti-tubulinandanti-HAepitopetagasdescribeinSection2(materialsandmethods).(C)HeLacellsweretransfectedwithempty vector,LPMV-V-HAorNipahV-V-HAexpressionplasmids.Beforefixation,cellsweretreatedfor30minwith1000U/mlofIFN␤.Cellswerefixed,permeabilized,andstained withantibodiesagainstSTAT1andHAasdescribedinSection2(materialsandmethods).(D)HeLacellsweretransfectedwithemptyvector,LPMV-V-HAorNipahV-V-HA expressionplasmids.Beforefixation,cellsweretreatedfor30minwith1000U/mlofIFN␤.Cellswerefixed,permeabilized,andstainedwithantibodiestoSTAT2andHA,as describedinSection2(materialsandmethods).NuclearDNAwasstainedwithDAPI.Imageswereobtainedusingconfocalmicroscopy.

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Fig.5. LPMV-VproteininteractswithhumanandporcineSTAT2.(AandB)LPMV-V-HA,NipahV-V-Ha,PIV5-V-Haplasmidsandemptyvectorweretransfectedin293T cells.At24hpostransfection,thecellsweretreated(orleftuntreated)with1000U/mlIFN-␤for24hat37◦_C.₄₈_h_after_transfection_cells_were_lysate._Aliquots_for_total proteinanalyseswereseparated,andtheremainingsupernatantswereincubatedfor3hwiththeRedAnti-HAafﬁnitygelantibodyorwithoutantibody.Thepelletswere washedsixtimeswithwhole-cellextractbufferandﬁnallydissolveddirectlyinLaemmlisamplebuffer.Thesampleswereanalyzedonsodiumdodecylsulfate(SDS)4–15% polyacrylamidegels.ProteinswerethentransferredtonitrocellulosemembranesandwereprobedwithantibodiesagainstSTAT1,STAT2,HAandActin.(C)Reciprocal co-immunoprecipitationin293Tcellsusingananti-STAT2antibodywasperformed.(D)Co-immunoprecipitationassaywasperformedinporcinecellsPK-15.

withPBS,blockingofnonspeciﬁcbindingsiteswasperformedin PBG(1XPBS,5%BSA,0.2%ﬁshgelatin)for30minatroom tem-perature.TheincubationwithprimaryantibodiesdilutedinPBG (anti-STAT1andanti-STAT2at1:100dilution,andanti-HAat1:500 dilution) wasperformed for 1h at 37◦C. Thenthe slides were washedinPBSandincubatedfor1hatroomtemperaturewith

thesecondaryantibodydilutedinPBGAlexaFluor555(Invitrogen) conjugatedtomouseimmunoglobulinGwasusedtovisualize HA-taggedVproteins,AlexaFluor488(Invitrogen)conjugatetorabbit immunoglobulinGwasusedtovisualizeeitherSTAT1orSTAT2. Nuclearchromatinstainingwasperformedbyincubationin block-ingsolutioncontaining0.5mg/ml4,6-diamidino-2-phenylindole,

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DAPI(Sigma–Aldrich).Cellswerewashedandcoverslipsmounted using Prolong antifade reagent (Invitrogen). Images were cap-turedusingaLeicaSP5-DMconfocalmicroscopeattheMicroscopy SharedResearchFacilityatIcahnSchoolofMedicineatMountSinai.

3. Results

3.1. LPMV-VproteinantagonizesIFN˛/ˇbutnotIFNsignaling pathway

The IFN antagonist function of the V proteins of several paramyxovirusesis wellcharacterized. In ordertoevaluatethe potentialofLPMV-Vprotein-mediatedinhibitionofIFN␣/␤-and IFN␥-mediatedsignaling,wefirstanalyzedtheexpressionand cel-lularlocalizationofHA-taggedLPMV-Vproteinin293TandHeLa cells,respectively.Westernblotanalysisshowedtheexpression andexpectedmolecularmassofapproximately42kDaofLPMV-V protein,whichiscloserinsizetoPIV5VproteinthanNipahV-V pro-tein(Fig.1A).Indirectimmunofluorescenceanalysisrevealedboth nuclearandcytoplasmiclocalizationofLPMV-Vprotein,andsolely cytoplasmiclocalizationofthecontrolproteinNipahV-V(Fig.1B). NextweexaminedtheeffectsofLPMV-VproteinontypeI IFN-ortypeIIIFN-mediatedgeneexpressionusingluciferasereporter genesunderthecontrolofIFN-responsivepromoters.293Tcells wereco-transfectedwithpCAGGSplasmids encodingNipahV-V, PIV5-VandLPMV-Vproteins,areporterplasmidpISRE54-CAT,a reporterplasmidpGAS-firefly-luciferaseandaplasmiddrivingthe constitutiveexpressionofrenilla-luciferaseusedasinternal con-trol.Twenty-fourhoursposttransfectionthecells weretreated with1000U/mlofIFN␤orIFN-␥and24hlatercellswerelysed andanalyzed forluciferase activity.Our resultsshowthatcells transfected with empty plasmid and treated with IFN showed a significantincrease in luciferaseexpression, asexpected. The cells expressing LPMV-V protein, showed significantly reduced luciferaseexpressiondrivenbytheISREpromoter,whichwas com-parabletothatseenwiththepositivecontrolsNipahV-VandPIV5-V proteins.Incontrast,LPMV-Vdidnotreduceluciferasegene expres-siondrivenbytheGASpromoter,whilebothNipahV-VandPIV5-V proteins,asexpected,wereabletodoso.(Fig.2AandB).These resultsindicatethatLPMV-VproteinspecificallyevadestypeIbut nottypeIIIFNsignaling.

ToexaminethebiologicalrelevanceofLPMV-VproteininIFN antagonism,weanalyzedtheeffectofLPMV-Vonreplicationof anIFN-sensitiveNewcastlediseasevirusthatexpressesGFP (NDV-GFP)inthepresenceoftypeIIFN(Parketal.,2003).PK13cellswere transfectedwithplasmidsencodingLMPV-VandPIV5-Vproteins. Twenty-fourhoursposttransfection,cellswereeithermocktreated ortreatedovernightwith1000U/mloftypeIIFN.Thefollowingday thecellswereinfectedwithNDV-GFP.Asexpected,incells trans-fectedwithemptyplasmidandtreatedwithIFN-␤,NDVwasunable toreplicateasevidencedbylackofGFPexpression(Fig.2C,panels 1and2).However,incellsexpressingLPMV-Vprotein,NDV-GFP replicationwasenhanced,similartothepositivecontrolPIV5-V proteinconﬁrmingthatLPMV-VproteinblockstypeIIFN medi-atedantiviralactivity(Fig.2C,panels 3and 4).However,when cellsweretransfectedwithLMPV-V-HAexpressionplasmid20h aftertypeIIFNtreatment,LMPV-Vproteinwasunabletorescue NDV-GFPinfectivity,indicatingthatwhileLMPV-Vproteininhibits typeIIFNsignaling,LMPV-Vproteindoesnotinhibitanalready establishtypeIIFN-inducedantiviralstate(Fig.2D).

3.2. LPMV-VproteindampenstheinductionoftypeI interferon-stimulatedgenesISG15andMXA.

TodetermineifLPMV-VproteininhibitsISGexpressionatthe RNAlevel,quantitativeRT-PCRwasusedtomeasureISG15and MXAgeneexpressionin293Tcellsthathadbeenstimulatedwith IFN␤for12h.ResultsshowedthatthecellsexpressingLPMV-V pro-teinhad25-and325-foldlowerISG15andMXAtranscriptlevels, respectively,thancellstransfectedwithanemptyplasmid, com-parabletothereductionshowedbythecontrolproteinsNipahV-V andPIV5-V(Fig.3AandB).

3.3. LPMV-VproteindoesnotreducetheinductionoftypeII interferon-stimulatedgenesIP10,IRF1

AquantitativeRT-PCRwasalsousedtomeasureIP10andIRF1 geneexpressionin293TcellsthathadbeenstimulatedwithIFN␥ for12h.ResultsshowedthatexpressionofLMPV-Vproteinhadno effectonIP10andRF1transcriptlevels(Fig.3CandD).

3.4. LPMV-VreducestypeIIFN-dependentphosphorylationof STAT2andSTAT1butnottypeIIIFN-dependentphosphorylation ofSTAT1

STAT1and STAT2arethetargetsofseveralParamyxovirusV proteins(Ramachandranand Horvath, 2009). Todeterminethe mechanism of LPMV-V protein inhibition of IFN signaling, we analyzed the effect of LPMV-V protein expression on the total levels of cellular STAT1 and STAT2 and the type I and type II IFN-inducedphosphorylationofSTAT1andSTAT2.Westernblot analysisshowedthatthetotalSTAT1andSTAT2levelsremained constantincellsexpressingLPMV-Vprotein,comparabletocells expressing an empty plasmid. However, expression of PIV5-V protein resulted in a loss of total STAT1, which is consistent withpublished reports (Didcocket al., 1999b) (Fig. 4A and B). ExaminationofLPMV-VproteineffectonthetypeIIFN-induced phosphorylation of STAT1 and STAT2 by western blotanalysis showedthat293TcellsexpressingLPMV-Vprotein,similarlyto those expressingNipahV-V andPIV5-Vproteins, had a reduced levelofIFN-inducedphosphorylationofSTAT1-Y701and STAT2-Y690.Thetransfectionefﬁciency(closeto90%)inthiscasecould explaintheresidual,phoshoSTAT1andSTAT2activityincellsthat expressLPMV-V,NipahV-V,andPIV5-Vproteins.Thisisincontrast towhatwasobservedincellstransfected withanemptyvector (Fig.4A).TheresultsindicatethatLPMVproteinhadnoeffecton thetotalcellularlevelsofSTAT1andSTAT2butinsteadinhibited thetypeIIFN-activatedphosphorylationofSTAT1andSTAT2. Dif-ferentresultswereobtainedwhenweanalyzedtheroleofLPMV-V proteinonthetypeIIIFN-inducedphosphorylationofSTAT1.Our datasuggestthatLPMV-Vproteinstilldoesnotinhibitefﬁciently theinductiontheSTAT1phosphorylationundergammainterferon stimulation(Fig.4B).

3.5. LPMV-VproteinpreventstypeIIFN-inducedSTAT1and STAT2nucleartranslocation

SincewehadobservedthattheLPMV-Vproteindidnotaffect thetotallevel ofSTAT1and STAT2but reducedthetype I IFN-inducedphosphorylationofSTAT1andSTAT2,wehypothesized that the STATs protein would be retained in the cytoplasm in thepresence of LPMV-V protein. Using confocal laser scanning microscopy,weshowedthatincellstransfected withanempty plasmidthentreatedwithtypeIIFN,STAT1andSTAT2translocate tothenucleus.However,theIFN-inducedtranslocationofSTAT1 andSTAT2wasinhibitedincellsexpressingLPMV-Vproteinsimilar tothepositivecontrolNipahV-Vprotein(Fig.4CandD).Thus

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LPMV-G.Pisanellietal./VirusResearch213(2016)11–22 19

Fig.6.Thelast18aminoacidsofLPMV-VproteinarenecessaryforbindingSTAT2.(A)AlignmentofVproteinsfromdifferentvirusintheParamyxovirusfamilyandschematic representationLPMV-V-HAC-Terminaldeletionsmutants(V-C18-HA,V-C25-HA,V-C50-HA,V-C75-HA).(B)Co-immunoprecipitationassayof293Tcellstransfected withLPMV-VHAdeletionsmutants(V-C18-HA,V-C25-HA,V-C50-HA,V-C75-HA)plasmidsandemptyvectorweretransfectedin293Tcells.At48hpostransfection, thecellswerelysate.Aliquotsfortotalproteinanalyseswereseparated,andtheremainingsupernatantswereincubatedfor3hwiththeRedAnti-HAaffinitygelantibodyor withoutantibody.Thepelletswerewashedsixtimeswithwhole-cellextractbufferanddissolveddirectlyinLaemmlisamplebuffer.Thesampleswereanalyzedonsodium dodecylsulfate(SDS)4–15%polyacrylamidegels.ProteinswerethentransferredtonitrocellulosemembranesandwereprobedwithantibodiesagainstSTAT1,STAT2,HAand Actin.(C)293Tcellswereco-transfectedwithpCAGGSplasmidsencodingNipahV-V,PIV5-VandLPMV-V-Ha,V-C18-HA,V-C25-HA,V-C50-HA,V-C75-HAproteins, areporterplasmidpISRE54-firefly-luciferaseandaplasmiddrivingtheconstitutiveexpressionofrenilla-luciferaseusedasinternalcontrol.24hposttransfectionthecells weretreatedwith1000U/mlofIFN␤and24hlatercellswerelysedandanalyzedforISRE54luciferaseactivity,normalizedwithrenillaluciferaseactivity.

Vprevents thetypeIIFN-induced phosphorylationandnuclear translocationofSTAT1andSTAT2,thuspreventingformationof theISGF3complexandthesubsequentbindingattheISREofISGs. 3.6. LPMV-VproteinbindsSTAT2protein

Since theV proteinof severalparamyxoviruses binds either STAT1 and STAT2 or both, we examined whether LPMV-V protein has the ability to bind STAT1 and STAT2 using

co-immunoprecipitation assays. 293T cells were transfected with LPMV-VHA,NipahV-V-HA,PIV5-VHAplasmidsandemptyvector HAtagged.Cellextractswereimmunoprecipitatedtaking advan-tageoftheHA epitope,andimmunecomplexeswereprocessed forimmunoblottingwithanti-STAT1andanti-STAT2 antibodies. OnlySTAT2proteinwasfoundintheLPMV-Vimmunecomplexes, whereasNipahV-Vprotein,aspredicted,pulleddownbothSTAT1 andSTAT2proteinsashavebeenpreviouslyreported(Fig.5A).

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ToruleoutthepossibilitythatlowlevelsofSTAT1was responsi-bleforthephenotypeofspecificbindingwithSTAT2,westimulated cells with IFN for 24h to increase the levels of STAT1, which is an ISG. We then performed a co-immunoprecipitation assay toevaluate whetherLPMV-V protein would beable to precip-itate STAT1 protein when higher levels of STAT1 are present. Theresultwassimilartowhatweobservedperformingthe co-immunoprecipitationintheabsenceofIFN␤nobindingwithSTAT1 wasdetected(Fig.5B).Wethenproceededtoconfirmourresultsby performingareciprocalco-immunoprecipitation.293Tcellswere mocktransfectedortransfectedwithplasmidsofinterest.Thecell extractswereimmunoprecipitatedwithSTAT2antibody,andthe immunecomplexeswereprobedforwithanti-HAantibody.The resultsconfirmedthatSTAT2bindsLPMV-Vprotein(Fig.5C).Since LPMVaffectspigs,wealsoexaminedwhetherLPMVVproteinbinds porcineSTAT2.Usingco-immunoprecipitationassaysinPK13cells, acelllineoriginatedfromswine,weshowthatLPMV-Vprotein bindsporcineSTAT2andnotSTAT1(Fig.5D).

In order todetermine theregion of LPMV-V protein that is requiredforitsinteractionwithSTAT2,wedecidedtofocusour attentionontheC-terminalregionthathasamain rolefor IFN antagonism in theParamyxoviridae family (Ramachandran and Horvath,2009). Four HA-tagged expression vectorswere engi-neered(Fig.6A).Oneconstructwithadeletionofthelast18amino acids(V-C18-HA),asecondconstructwithadeletionofthelast 25aminoacids(V-C25-HA),athirdconstructwithadeletionof thelast50aminoacids(V-C50-HA)andafourthconstructwitha deletionofthelast75aminoacids(V-C75-HA)wereengineered andtestedinaco-immunoprecipitationassayinPK13cellsasin

Fig.6panelA.Theconstructthathadadeletionofthelast18amino acidsdidnotbindSTAT2proteinindicatingthattheC-terminusof LPMV-VproteiniscriticalforitsbindingtoSTAT2(Fig.6B).Next weanalyzedtheabilityofthemutantproteinstoinhibitthetype IIFNsignalingcascade usinga reportergene assay.Ourresults showthatcellsexpressingtheLPMV-Vproteinlackingthelast18 aminoacidswereunabletoinhibitIFNsignalingsimilarto mock-transfectedcellsthatweretreatedwithIFN(Fig.6C).Theseresults areconsistentwiththoseobtainedintheco-immunoprecipitation experimentwiththeLPMV-Vproteinsdeletionmutant,conﬁrming thatthelast18aminoacidsarecriticalforthebindingof LPMV-VproteintoSTAT2andforLPMVVproteintypeIIFNantagonist activity.

4. Discussion

TheabilityofparamyxovirusestosubverttheIFNresponseis wellconserved,withtheonlyexceptionbeingHuPIV4(Goodbourn and Randall, 2009; Nishio et al., 2005b; Ramachandran and Horvath,2009).Thischaracteristic is due mainly totheV pro-teins.IntheHenipavirusgenus,theNipahvirusandHendravirus Vproteins,for example,inhibitIFNresponsesbyforming high-molecular-weight complexes with both STAT1 and STAT2 and preventingSTATsphosphorylationand translocation(Rodriguez etal.,2002,2003).TheVproteinofmeaslesvirus,ofthe Morbil-livirusgenus,blocksIFN-inducedSTAT1andSTAT2nuclearimport withoutdegradingSTATsandpreventingSTATsphosphorylation (Takeuchietal.,2003).STATpolyubiquitinationand proteasome-dependentdegradationisthemechanismbywhichtheVproteins ofRubulavirusmembers,likehumanparainﬂuenzavirus5,human parainﬂuenza virus 2 and mumps virus inhibit IFN signaling (Andrejevaetal.,2002;Didcocketal.,1999b;Kubotaetal.,2005; Nishioetal.,2002,2005a;Parisienetal.,2001,2002;Ulaneand Horvath,2002)withtheexceptionofMauperavirusthatblocks IFNsignalingwithoutSTATsdegradation(Hagmaieretal.,2007).

InthisstudyweshowthatLPMV-VproteinevadestypeI inter-feronsignalingbutnottypeII(Fig.2AandB).Adrasticreduction ofISG15andMXAtranscriptlevelswasfoundincellswith LPMV-VproteinexpressionstimulatedwithtypeIIFN,comparedtothe mock-transfectedcells (Fig.3Aand B)which is consistentwith publishedreports(Flores-OcelotlMdeletal.,2011).Incontrast, cellsexpressingLPMV-Vprotein,typeIIIFNstimulationdoesnot reduceIP10andIRF1mRNAlevels(Fig.3CandD).Thebiological impactofLPMV-VproteinintypeIIFNantagonismwasconﬁrmed byitscapacitytorescueNDV-GFPreplicationinthepresenceof IFN(Fig.2Cpanel4).WeshowthatLPMV-Vproteindoesnotaffect thetotallevel ofSTAT1and STAT2,butinhibitsthetype I IFN-inducedphosphorylationandnucleartranslocationofbothSTAT1 andSTAT2.However,LMPV-VproteindoesnotinhibitthetypeII IFN-inducedphosphorylationofSTAT1(Fig.4A–D).Furthermore, weshowthatLPMV-VproteinbindsselectivelytoSTAT2inhuman andporcinecells(Fig.5A–D)andthatthelast18aminoacidsof LPMVVarerequiredforbindingSTAT2.(Fig.6BandC)

TakentogetherthesedatashowthattheLPMV-Vprotein antag-onizesthetypeIIFNresponsebutnotthetypeIIwithouttargeting STAT1andSTAT2proteinsfordegradation.ThiscontrastswithV proteinsfromothermembersoftheRubulavirusgenus,suchas PIV5,mumpsorHuPI2,whichantagonizetypeIIFNsignalingby inducingdegradationoftheSTAT1andSTAT2proteins(Didcock et al.,1999a,b; Kubotaet al.,2005; Parisienet al., 2001,2002; Preciousetal.,2005a,b;UlaneandHorvath,2002).LPMV-V pro-teinIFNevasionstrategypartiallyresemblesthatofHenipavirus Vprotein,withitsselectivebindingwithSTAT2protein,though notwithSTAT1,andwiththeinhibitionofSTATphosphorylation andsubsequentnucleartranslocation(Rodriguezetal.,2002,2003). However,LPMV-VproteindoesnotaffectthetypeIIinterferon sig-nalingpathwaydespiteitsinhibitionoftypeIinterferon-mediated STAT1 phosphorylation. The IFN signaling antagonism strategy usedbytheVproteinofmeasles,aMorbillivirusgenusmember, ismostsimilartothatusedbyLPMV-Vprotein.Infactmeasles Vproteininhibitstype IIFNbutnottypeIIbyinhibitingtypeI IFN-mediatedSTAT1andSTAT2phosphorylation.(Takeuchietal., 2003).ThesedatasuggestthatbytargetingSTAT2,LPMV-V pro-teininhibitsbothSTAT2andSTAT1phosphorylationinresponseto typeIIFNsignaling,resultingininhibitionofSTATsproteinnuclear translocation.Thesedataareconsistentwithwhatisdescribedin theliteratureontheprimaryrolethatSTAT2proteinhasinthetype Isignalingactivation(Leungetal.,1995;Lietal.,1997;Qureshi etal.,1996).SimilartothestrategyusedbyLPMV-Vproteinto evadetheIFNsignalingcascadeintheRubulavirusgenus,isthe oneadoptedbyMauperavirusVprotein.BothinhibitIFNsignaling withoutdegradingSTATs.WhileMauperavirusVproteininhibits IFNsignalingbybindingbothSTAT1andSTAT2and preventing theirnucleartranslocation,withoutaffectingtheir phosphoryla-tion(Hagmaieretal.,2007),LPMV-V proteinantagonizestypeI IFNsignalingcascadebybindingspeciﬁcallytoSTAT2,preventing STATsnucleartranslocationandaffectingtheirphosphorylation.

AcrosstheParamyxovirusfamily,VproteinsevadetheIFN sig-nalingpathwayusingavarietyofstrategies(Andrejevaetal.,2004; Didcock et al.,1999a,b; Kubotaet al., 2005; Motzet al., 2013; Nishioetal.,2002,2005a;Parisienetal.,2001,2002;Preciousetal., 2005b; Rodriguezet al.,2002,2003).Our studyhasfoundthat LPMV-Vprotein’sIFNsignalingevasionstrategydiffersfromthe proteasome-mediateddegradationstrategythatisnormallyused byVproteinsfrommembersofitsgenus,Rubulavirus.Thisisdespite thefactthattheC-terminaldomainofLPMV-Vproteinissimilarto thatoftheotherVproteinsintheRubulavirusgenusandthatithas eightcysteineresidues,ofwhichsevenareconservedinallV pro-teins(Bergetal.,1992)(Fig.5A).ThesimilarC-terminusregionof theLPMV-VproteinwiththeC-terminusofotherparamyxovirusV proteinsislikelytoberelatedtotheiressentialroleininteracting

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withMDA-5,(Davisetal.,2014;Motzetal.,2013;Rodriguezand Horvath,2014)however,thelast21aminoacidsofLPMV-Vprotein isunique,andthiscouldhaveakeyroleintheIFNsignalingevasion mediatedbySTAT2inactivation.Thisislikelythecaseaswefound thatthelast18aminoacidsofLPMV-VarecriticalforSTAT2 bind-ingandtypeIIFNsignalinginhibition.Althoughourresultsstill needtobeconﬁrmedinthecontextofLPMVinfection,this infor-mationcouldbeofuseinthefuturewhendesigningLPMVvaccine candidatesorantiviraltherapeuticsagainstLPMV.

Acknowledgments

These studies were partly supported by NIAID grant U19AI083025 (to AG-S) and by a National Institute of Health Fellowship (to ML-R). Confocal laser scanning microscopy was performedatISMMS-MicroscopySharedResourcefacility.

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