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1、JournalofVisualizedExperimentswww.jove.comVideoArticleProductionofPseudotypedParticlestoStudyHighlyPathogenicCoronavirusesinaBiosafetyLevel2Setting1,23343,531JeanK.Millet,TiffanyTang,LakshmiNathan,JavierA.Jaimes,Hung-LunHsu,SusanDaniel,GaryR.Whittaker1DepartmentofMi
2、crobiologyandImmunology,CollegeofVeterinaryMedicine,CornellUniversity2INRA,VirologieetImmunologieMoléculaires3RobertFrederickSmithSchoolofChemicalandBiomolecularEngineering,CornellUniversity4DepartmentofMicrobiology,CollegeofAgriculturalandLifeSciences,CornellUniver
3、sity5HoraeGeneTherapyCenter,UniversityofMassachusettsMedicalSchoolCorrespondenceto:GaryR.Whittakeratgary.whittaker@cornell.eduURL:https://www.jove.com/video/59010DOI:doi:10.3791/59010Keywords:CancerResearch,Issue145,Pseudotypedparticle,pseudovirion,coronavirus,CoV,s
4、pikeprotein,severeacuterespiratorysyndromecoronavirus,SARS-CoV,MiddleEastrespiratorysyndromecoronavirus,MERS-CoV,murineleukemiavirus,MLVDatePublished:3/1/2019Citation:Millet,J.K.,Tang,T.,Nathan,L.,Jaimes,J.A.,Hsu,H.L.,Daniel,S.,Whittaker,G.R.ProductionofPseudotypedP
5、articlestoStudyHighlyPathogenicCoronavirusesinaBiosafetyLevel2Setting.J.Vis.Exp.(145),e59010,doi:10.3791/59010(2019).AbstractTheprotocolaimstogeneratecoronavirus(CoV)spike(S)fusionproteinpseudotypedparticleswithamurineleukemiavirus(MLV)coreandluciferasereporter,usin
6、gasimpletransfectionprocedureofthewidelyavailableHEK-293Tcellline.Onceformedandreleasedfromproducercells,thesepseudovirionsincorporatealuciferasereportergene.Sincetheyonlycontaintheheterologouscoronavirusspikeproteinontheirsurface,theparticlesbehaveliketheirnativeco
7、ronaviruscounterpartsforentrysteps.Assuch,theyaretheexcellentsurrogatesofnativevirionsforstudyingviralentryintohostcells.Uponsuccessfulentryandinfectionintotargetcells,theluciferasereportergetsintegratedintothehostcellgenomeandisexpressed.Usingasimpleluciferaseassay
8、,transducedcellscanbeeasilyquantified.Animportantadvantageoftheprocedureisthatitcanbeperformedinbiosafetylevel2(BSL-2)facilitiesinsteadofB
