DevelopmentalandComparativeImmunology53(2015)293e302

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DevelopmentalandComparativeImmunology

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Identi?cationandfunctionalcharacterizationofviperinofamphioxusBranchiostomajaponicum:Implicationsforancientoriginofviperin-mediatedantiviralresponse

MiaomiaoLei,HaohanLiu,ShoushengLiu,YuZhang**,ShicuiZhang*

LaboratoryforEvolution&Development,InstituteofEvolution&MarineBiodiversityandDepartmentofMarineBiology,OceanUniversityofChina,Qingdao266003,PRChina

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Articlehistory:

Received19April2015Receivedinrevisedform9July2015

Accepted11July2015

Availableonline17July2015Keywords:Amphioxus

CephalochordateViperinInterferons

Antiviralactivity

abstract

Viperin,anantiviralprotein,hasbeenshowntobeactiveagainstawiderangeofDNAandRNAviruses,butnoinformationisavailableregardingfunctionalcharacterizationofviperinininvertebratespecies.Inthisstudy,weclearlydemonstratethatamphioxus(Branchiostomajaponicum)viperin,BjVip,hasfeaturesincommonwiththoseofvertebrateviperin,includingthepresenceoftheSAMsuperfamilydomainwiththecharacteristicCNYKCGFCmotif,syntenicconservation,andpredicted3Dstructure.Bjvipexhibitsatissue-speci?cexpressionwithabundantlevelsinthehepaticcecum,hind-gut,gillandmuscle,andfollowingchallengewiththeviralmimicpolyI:C,itsexpressionissigni?cantlyup-regulated,suggestinganinvolvementofBjVipinimmuneresponseofamphioxusagainstviralinfection.Importantly,weshowthatthecellstransfectedwithBjvipisabletokillLCDVorinhibitingitspropagation,andco-incubationofrBjVipwithWSSVmarkedlyattenuatesitsinfectivity.Thus,weprovidethe?rstevidencesthatamphi-oxusviperin,likethatofvertebrates,iscapableofpromotingresistanceagainstviralinfectioninvitroandinvivo,indicatingthatviperin-mediatedantiviralresponsealreadyemergedintheprimitivechordate.Wealsoprovethatamphioxusviperinhasevolvedunderpositiveselection.

?2015ElsevierLtd.Allrightsreserved.

1.Introduction

Vertebrateinterferon(IFNs)proteins,mainlycomprisingtypeI(IFN-a/b),type-II(IFN-g)andtypeIII(IFN-l)IFNs,arerapidlyinducedfollowingviralinfectionandsubsequentlyactivatethou-sandsofinterferon-regulatedgenes(IRGs)orinterferon-stimulatedgenes(ISGs)(DeVeeretal.,2001;SenandSarkar,2007).ISGsencodeproteinsthatgenerallydisplayarangeofantiviralactivitiesagainstdiversepathogens(SadlerandWilliams,2008;Sen,2001).Viperin(virusinhibitoryprotein,endoplasmicreticulum-associ-ated,interferon-inducible)isoneofISGs-encodingproteins,whichcouldinhibitmanyDNAandRNAvirusessuchasCHIKV,HCMV,HCV,DENV,WNV,SINV,in?uenza,andHIVLAIstrainetc.Initiallyidenti?edascig5inHCMV-infectedprimaryhumanforeskincells

*Correspondingauthor.Room205,KeXueGuan,5YushanRoad,OceanUni-versityofChina,Qingdao266003,PRChina.

**Correspondingauthor.Room319,DarwinBuilding,5YushanRoad,OceanUniversityofChina,Qingdao266003,PRChina.

E-mailaddresses:yuzhang@ouc.edu.cn(Y.Zhang),sczhang@ouc.edu.cn(S.

Zhang).

http://dx.doi.org/10.1016/j.dci.2015.07.008

0145-305X/?2015ElsevierLtd.Allrightsreserved.

(Zhuetal.,1997),viperinissubsequentlyfoundinrainbowtroutasthevig1geneinduciblebyviralhaemorrhagicsepticaemiavirusandinmouseasthevig1/cig5homologueinduciblebyvesicularstomatitisvirusandpseudorabiesvirus(Boudinotetal.,1999,2000).Viperinhasnowbeenidenti?edinvariousspeciesofver-tebrates.Allviperinhomologuesarehighlyconservedinthepri-marysequence,whichincludesarelativelylessconservedN-terminalamphipathica-helixrequiredforendoplasmicreticulum(ER)localization(HinsonandCresswell,2009a,b),acentralradicalS-adenosylmethionine(SAM)domainwhichcontainsaCxxxCxxCmotifimportantfortheformationofa[4Fee4S]cluster(Freyetal.,2008),andahighlyconservedC-terminalregionthatisknowntobecriticalforantiviralactivityagainstanumberofviruses(HelbigandBeard,2014;Helbigetal.,2013).PositiveselectionhasbeenreportedtodriverapidevolutionofcertainaminoacidresiduesintheN-terminalregionsofthehighlyconservedviperinproteinsof?shes(Padhi,2013).

Viperincouldbeinducedinavarietyofcelltypesbydifferentcellularfactors,suchastypeI,IIandIIIIFNs,DNAandRNAviralproteins,viralmimicpolyriboinosinicpolyribocytidylicacid(polyI:C)andpolysaccharide(Boudinotetal.,1999;ChinandCresswell,

294M.Leietal./DevelopmentalandComparativeImmunology53(2015)293e302

2001;Grewaletal.,2000;Zhouetal.,2007;Zhuetal.,1997).Interestingly,theactivationofviperinmRNAaccumulationbyHCMVdoesnotrequiretheactionofIFNsandexpressionofviralgenes(Zhuetal.,1997),suggestingthatthereisIFN-independentpathwayforinductionofviperin.Therefore,vertebrateviperincouldbeinducedineitherIFN-dependentorIFN-independentpathway.ViperinappearstouseavarietyofdifferentantiviralmechanismsfrominteractingdirectlywithviralandhostproteinsessentialforviralreplicationtointeractionwithhostorganellessuchastheER,lipiddropletsandmitochondria(BlasiusandBeutler,2010;Gaoetal.,2004;Jiangetal.,2008;Reizisetal.,2011;Saitohetal.,2011;Wangetal.,2012,2007).

Viperinhomologueshavealsobeenpredictedinavarietyofinvertebratespecies,includingsponge,daphnia,insectandamphioxus(Padhi,2013).However,uptonow,functionalcharac-terizationofviperinhasbeenexclusivelyrestrictedtovertebrateorganisms;littleisknownaboutthefunctionandevolutionofinvertebrateviperin,althoughitwasshownthatviperingeneexpressionwasup-regulatedinresponsetopolyI:CinPaci?coyster(Greenetal.,2014).Inaddition,asinvertebratesingeneralandamphioxusinparticularlackIFNs,whetherviperinofinvertebrates,likethatofvertebrates,playsdirectantiviralrolesremainsun-known.Amphioxus,theextantsurvivorofanancientchordatelineage,isthebestavailablestand-infortheproximateinvertebrateancestorofvertebratesandthusanidealmodelforgaininginsightsintotheoriginandevolutionofvertebrates(Stach,2008;Zhangetal.,2009).Theaimsofthisstudywere,therefore,tocloneviperinhomologuefromtheamphioxusBranchiostomajaponicum,toexamineitsevolutionandexpression,andtoinvestigateitsantiviralactivity.

2.Materialsandmethods

2.1.CloningandsequenceanalysisofamphioxusviperincDNAAdultamphioxusB.JaponicumwerecollectedduringthebreedingseasoninthevicinityofQingdao,Chinaandculturedinaeratedseawaterforoneweekbeforeexperimentsatroomtem-perature.TotalRNAswereextractedwithTrizol(Invitrogen)fromB.japonicumaccordingtothemanufacturer'sinstructions.AfterdigestionwithRNase-freeDNase(TaKaRa)toeliminatethegenomiccontamination,the?rst-strandcDNAwassynthesizedwithreversetranscriptionsystem(Promega)usingoligod(T)primer,andusedasPCRtemplate.Thefragmentoftheviperingene,namedBjvipthereafter,wasampli?edbyPCRwiththeprimerpairsP1eFandP1eR(Table1)thatweredesignedusingPrimerPremier5.0programonthebasisofviperinsequencesidenti?edinBran-chiostoma?oridaegenomedatabase()andBranchiostomabelcherigenomedatabase().Athree-dimensional(3D)modelingofBjVipwaspredictedbyfully-automatedproteinstructurehomologymodeling(http://

Table1

Sequencesoftheprimersusedinthisstudy.PrimerP1eFP1eRP2eFP3eFP4eRP5eRP6eFP6eRP7eFP7eRP8eFP8eRP9eFP9eRP10eFP10eRP11eFP11eRP12eFP12eRP13eFP13eRP14eFP14eR

Primersequence(50e30)

TCAGGTTCTGTAAGKDSGAGTTCGAGATGGTTCCTTCCGT

TGGCGTTCAAGATAAACTCGGAGCGTCAGCATAGTCAGCAACGTTGCTGACTATGCTGACGCTCCCGAGTTTATCTTGAACGCCACACCGCCAAGAAAGGGCTGAGGGCTGCCGTTGCTGACTATGCTTCCAGCCTTCATTCCTCGCGGTGTTGGCGTACAGGTC

CGGAATTCATGCTGATTGTGACGTGTCCGCTCGAGCCAGTCCAACTTTAAGTC

CGGGGTACCATGGTGAGCAAGGGCGAGGAG

CGCGGATCCGATTACTTGTACAGCTCGTCCCATGCCAGGTACAAACAGCACCTAAACATGCACCGTCAAAGATTACAGGAGAAGCCCATCTACGAGGGCTACGCTCTCGGCTGTGGTGGTGAAG

GGAATTCCATATGATAATATTCCCGAAAGTCAAGAAGTCGGAATTCTTACCAGTCCAACTTTAAGTCAGCCAGCTCCAACACCTCCTCCTTCATTACTCGGTCTCAGTGCCAGAAGTAGCCGCCCTGGTTGTAGACTTCTCCATGTCGTCCCAGT

Primerampli?cationef?ciencies(%)

Ampliconlength(bp)55939571

97.6999.06

1691091071723

94.0898.15

1731271002

97.2494.08

162240

M.Leietal./DevelopmentalandComparativeImmunology53(2015)293e302295

swissmodel.expasy.org/)usinghumanviperinandzebra?shviperinascomparison.

ThephylogenetictreewasconstructedintheJTTmodelofMEGA6.0usingtheaminoacid-basedmaximumlikelihood(ML)algo-rithm.Thereliabilityofeachnodewasestimatedbybootstrappingwith1000replications.Syntenicanalysisofviperinhomologuesofhuman,chimpanzee,mouse,platypus,chicken,zebra?nch,anolelizard,frog,zebra?sh,tilapia,elephantsharkandFloridaamphi-oxuswereobtainedfromtheSequenceViewer(http://parisonofM0andM3,andcomparisonofM7andM8.CodonsiteswithBayesEmpiricalBays(BEB)posteriorprobability!0.95wereconsideredtobeunderpositiveselection.Then,thepositiveselectionwasfurtherevaluatedwithbranch-speci?cmodel.Thismodelallowedustoexplorethedivergenceofdifferentbranchesinthephylogenyandcomputetheuratioofdifferentbranches(Yang,1998;YangandNielsen,1998).LRTswerecalculatedfromcomparisonofthefree-ratiomodelwiththeone-ratiomodel,andcomparisonoftwo-ratiomodelwiththeone-ratiomodel,todeterminewhetheruratioisidenticalamongdifferentbranches.2.2.Quantitativereal-timePCR(qRT-PCR)