mBio0017110s1Word文档格式.docx

mBio0017110s1Word文档格式.docx

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GenerationofrecombinantMHVvectorsandHCoV229E

ThegenerationofrecombinantMHVvectorsandrecombinantHCoV229EisbasedonreversegeneticsystemsestablishedforMHV-A59andHCoV-229E,respectively（1,2）.cDNAsencodingforrecombinantMHV-A59andHCoV229EareclonedintothevacciniavirusvNotI/tkandthecorrespondingrecombinantvacciniavirusesaredesignatedvMHV-inf-1andvHCoV-inf-1.TomodifytheclonedMHV-A59andHCoV229EcDNAs,therecombinantvacciniavirusesvMHV-inf-1andvHCoV-inf-1weremodifiedasdescribedbelowbyvacciniavirus-mediatedhomologousrecombinationusingtheE.coliguanosin-phosphoribosyltransferase（gpt）asselectionmarker（3）.TheresultingrecombinantMHVvectorcDNAsandrecombinantHCoV229EcDNAswereverifiedbyDNAsequencinganalysis.RecombinantMHVvectorRNAsandrecombinantHCoV229ERNAsweregeneratedbyT7bacteriophageRNApolymerase-mediatedinvitrotranscriptionusingrecombinantMHVvectorcDNAorrecombinantHCoV229EcDNAastemplate.TransfectionoftheresultingRNAsintoBHK-21cellsthatexpresstheMHV-A59nucleocapsidgene（forMHV-basedvectors）orHCoV229Enucleocapsidgene（forrecombinantHCoV229E）,gaverisetothereleaseofinfectiousparticlesintothecellculturesupernatant（3）.

ConstructionofrecombinantMHVvectorcDNAs

ToconstructtherecombinantvacciniavirusesvMHV-GP,vMHV-MelA,vMHV-GM/GPandvMHV-GM/MelA,multipleroundsofvacciniavirus-mediatedrecombinationweredoneinthefollowingorder.First,therecombinantvacciniavirusvMHV-nsp1Δ99（containingthefulllengthMHV-A59cDNAexceptfora99nucleotidedeletionwithinthensp1-codingregion;

（4））wasrecombinedwiththeplasmidpMHV-rec-3.TheplasmidpMHV-rec-3isbasedontheplasmidpGPT-1（5）andencodesMHVnucleotides（nts）27386–27965,thegptgeneandMHVnts28929–29655.Theresultinggpt+recombinantvacciniaviruswasrecombinedwithplasmidpRec3-GP-EGFPorpRec3-EGFP-MelA.TheplasmidpRec3-GP-EGFPisaderivateofpMHV-rec-3wherethegptgenewasreplacedbyageneencodingforafusionproteinofEGFPwiththeLCMVglycoproteinCTLepitopegp33-41（GP-EGFP）（6）.TheplasmidpRec3-EGFP-MelAisaderivateofpMHV-rec-3wherethegptgenewasreplacedbyageneencodingforafusionproteinofEGFPwiththeyeastubiquitin-codingsequenceandtheMel-A26-35A27LanalogpeptidederivedfromthehumanMelan-A/MART-1protein（EGFP-MelA）（7）.ThesecloningstepsreplacedtheMHVaccessorygenes4,5a,andtheMHVstructuralgeneEbythegenesencodingtheEGFPfusionproteinsGP-EGFPorEGFP-MelAintheresultingrecombinantvacciniavirusesvRec3-GP-EGFPandvRec3-EGFP-MelA,respectively.ToobtaintherecombinantvacciniavirusesvMHV-GPandvMHV-MelA,theMHVaccessorygenesNS2（encodingforaputativecyclicphosphodiesterase）,andHE（encodingforatruncatedhemagglutinin-esterase）weredeletedbytwoadditionalroundsofrecombinationoftherecombinantvacciniavirusesvRec3-GP-EGFPorvRec3-EGFP-MelAwiththeplasmidpRec2-gpt（containingMHVnts21326–21771,thegptgeneandMHVnts23887–24452）,followedbyasecondrecombinationoftheresultinggpt+cloneswiththeplasmidpRec2-Del（containingMHVnts21326–21771and23930–24452）.ToconstructtherecombinantvacciniavirusesvMHV-GM/GPandvMHV-GM/MelA,thesecondrecombinationwasdonewithplasmidpRec2-mGM-CSFthatcontainedthecodingsequenceofthemurineGM-CSFgene（GenBankaccessionnumber:

NM_009969）inordertoreplaceMHVaccessorygenesNS2andHE.ThestructureoftheMHVvectorsencodedintherecombinantvacciniavirusesvMHV-GP,vMHV-MelA,vMHV-GM/GPandvMHV-GM/MelAisillustratedinfigure1A.

ToconstructtherecombinantvacciniavirusesvHCoV-GP-EGFPandvHCoV-MelA,vacciniavirusvHCoV-inf-1containingthefull-lengthHCoV-229EcDNA

（2）wasrecombinedwiththeplasmidpHCoV-DCrec1.TheplasmidpHCoV-DCrec1isbasedonpGPT-1andcontainstheHCoV229Ents23634–24090,thegptgene,andHCoV229Ents24561–25030.Theresultinggpt+vacciniavirusclonewasthenusedtorecombinewithplasmidpHCoV-GP-EGFPorpHCoV-MelAinordertoreplaceHCoV229Eaccessorygene4bygenesencodingtheEGFPfusionproteinsGP-EGFPorEGFP-MelA.TheplasmidspHCoV-MelAandpHCoV-GP-EGFParederivatesoftheplasmidpHCoV-DCrec1wherethegptgenewasreplacedbytheEGFPfusionproteinsGP-EGFPorEGFP-MelA,respectively.ThestructureoftherecombinantHCoVsencodedintherecombinantvacciniavirusesvHCoV-GP-EGFPandvHCoV-MelAisillustratedinfigure7A.

RescueofrecombinantMHV-basedvectorsandhumancoronaviruses

TorescuerecombinantMHV-basedvectors,theMHVvector-encodingvacciniavirusDNAswereusedastemplateforinvitrotranscriptionwithT7bacteriophageRNApolymeraseasdescribed（3）.TheresultingMHV-basedvectorRNAswereelectroproratedintoBHK-MHV-NcellsthatexpresstheMHV-A59nucleocapsidgeneunderthecontroloftheTet/ONsystemasdescribed（3）.FollowingelectroporationtheBHK-MHV-Ncellsweremixedina1:

4ratiowith17clone-1cellsthathavebeenmodifiedtoexpresstheMHV-A59envelope（E）geneunderthecontroloftheTet/OFFsystem（17Eclone20packagingcells）（8）.Alternatively,theinvitrotranscribedMHV-basedvectorRNAshavebeentransfecteddirectlyinto17clone1-EM13cells（9）or17Eclone20packagingcells.24–72hposttransfectionthecellculturesupernatantcontainingMHV-basedvectorparticleswasharvestedandtheMHV-basedvectorswereamplifiedby1-5passageson17Eclone20packagingcellstoobtaintitersofapproximately1×

107pfu/ml.TheresultingMHVvectorstockswereverifiedfortheirintegritybyRT-PCR,sequencinganalysisandtransgeneexpression.

TorescuetherecombinantHCoV-GP-EGFPandHCoV-MelA,therecombinantvacciniavirusesvHCoV-GP-EGFPorvHCoV-MelAwereusedastemplateforinvitrotranscriptionwithT7bacteriophageRNApolymeraseasdescribed（3）.TheresultingrecombinantHCoV-GP-EGFPandHCoV-MelARNAswereelectroproratedintoBHK-HCoV-NcellsthatexpresstheHCoV-229EnucleocapsidgeneunderthecontroloftheTet/ONsystemasdescribed（3）.FollowingelectroporationtheBHK-HCoV-Ncellsweremixedina1:

4ratiowithhumanHuh7cells.24–72hposttransfectionthecellculturesupernatantcontainingrecombinantHCoV-GP-EGFPorHCoV-MelAwasharvestedandtherecombinantviruseswerefurtheramplifiedandtitratedonhumanHuh7cells.

Determinationofantiviralprotection

LCMV-WEstrainwaspropagatedonL929cells,andtiteredonMC-57cells.VacciniavirusesweregrownandtitratedonBSC40cells.Toexamineanti-viralprotection,naï

veB6micewereimmunizedwithMHV-GP,orMHV-GM/GPdilutedincoldBSS.Attheindicateddayspostimmunization（p.i）,micewerechallengedwith200pfuLCMV-WEi.v.orwith2×

106pfuofrecombinantvacciniavirusencodingeithertheglycoproteinofLCMV-WE,ortheglycoproteinofVSV.Fourdayspostchallenge,micewerekilledandvirustitersinthespleensweredeterminedbyLCMVinfectiousfocusassayonMC57cells.Fordeterminationofvacciniavirustiters,ovarieswerecollectedonday5postchallenge,andplaqueassaywasperformedasdescribedpreviously（10）.

Isolationofdendriticcellsandmacrophages,flowcytometry,immunofluorescence

Bonemarrow–derivedcDCsweregeneratedbyculturingerythrocyte-depletedbonemarrowcellsfor6to7daysinthepresenceofGM-CSFcontainingsupernatantfromthecelllineX63-GM-CSF（kindlyprovidedbyAntoniusRolink,UniversityofBasel,Basel,Switzerland）asdescribedpreviously（10）.cDCswerefurtherpurifiedusingOptiprepdensitygradientcentrifugation.Thioglycolate-elicitedmacrophageswerecollectedfromtheperitonealcavityofmiceandculturedovernightat37°

C.Non-adherentcellswereremovedbywashingwithice-coldPBS.SplenicDCswereisolatedfromsplenocytesuspensionsofcollagenasetypeII-digested（Gibco,InvitrogenBasel,Switzerland）spleens.TheCD11c+DCfractionwasenrichedusingmouseCD11c（N418）microbeads（MiltenyiBiotec）asrecommendedbythemanufacturer.

SplenocyteswereobtainedfromspleensofB6micefollowingdigestionwithcollagenasetypeIIfor20minat37°

CandresuspendedinRPMI/5%FCS.Forisolationofthelowdensityenrichedpopulation,cellswereresuspendedinPBSsupplementedwith2%FCS,2mMEDTAandoverlaidon20%Optiprepdensitygradientmedium（Sigma-AldrichCo.Basel,Switzerland）.Aftercentrifugationat700gfor15min,lowdensitycellswererecoveredfromtheinterfaceandresuspendedinRPMI/5%FCS.CellswerestainedwithdifferentlineagemarkersandanalyzedforEGFPexpressionwithaFACSCaliburflowcytometerusingtheCellQuestsoftware（BDBiosciences）.AntibodiesusedinthisstudywerepurchasedfromBDBiosciencesPharmingen（CD11c-PE,CD8α-PERCP）,Biolegend（CD19-PE,B220-APC,CD40-APC,CD11b-APC,CD86-APC）,andeBiosciences（F4/80-PE）.

Tetrameranalysisandintracellularcytokinestaining

PeptidespecificCD8+TcellsandexvivoproductionofIFN-γorTNF-αweredeterminedbytetramerstainingandintracellularcytokinestainingwereperformedasdescribedpreviously（4,11）.OrganswereremovedattheindicatedtimepointsfollowingimmunizationwithMHV-basedvectors.Tetramersweresynthesizedandappliedforstainingofbloodandspleensamplesaspreviouslydescribed（7,12）.CD8-APC（eBiosciences）andCD62L-FITC（MiltenyiBiotec）wereusedforthestaining.Forintracellularcytokinestaining,singlecellsuspensionsof106splenocyteswereincubatedfor5hat37°

Cin96-wellround-bottomplatesin200lculturemediumcontaining25U/mlIL-2and5g/mlBrefeldinA（Sigma）.Cellswerestimulatedwithphorbolmyristateacetate（PMA,50ng/ml）andionomycin（500ng/ml）（bothpurchasedfromSigma,Buchs,Switzerland）aspos