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1、Proc.Nati.Acad.Sci.USAVol.87,pp.6944-6948,September1990CellBiologyTheElglycoproteinofanaviancoronavirusistargetedtothecisGolgicomplex(intracellularproteintrafficking)CAROLYNE.MACHAMER*t,SUEANNMENTONEt,JOHNK.ROSE*,ANDMARILYNG.FARQUHARt§Departmentsof*PathologyandtCellBiology,YaleUniversitySchoolo
2、fMedicine,NewHaven,CT06510ContributedbyMarilynG.Farquhar,June20,1990ABSTRACTItwaspreviouslyreportedthattheElproteinMATERIALSANDMETHODSofanaviancoronaviruswastargetedtothejuxtanuclearregionCellsandViruses.AtT-20andCOS-1cellsweremaintainedinCOScellsexpressingtheproteinfromclonedcDNA,sug-inDulbecc
3、o'smodifiedEagle'smediumwith5%fetalbovinegestingthattheproteincontainsinformationfortargetingtoserum.Thegenerationofrecombinantvacciniavirusesen-theGolgicomplex.Thefirstofthreemembrane-spanningcodingthewild-typeElproteinandthemutantAml,2wasdomainswasrequiredforintracellulartargeting,becauseapre
4、viouslydescribed(5).mutantEl(Aml,2)lackingthisdomainwasdeliveredtotheAnti-ElAntibody.Thepolyclonalantibodyrecognizingtheplasmamembrane.WehaveusedimmunoelectronmicroscopyIBVElproteinwasgeneratedinarabbitimmunizedwithatolocalizethewild-typeElproteinwithinGolgielementsofsyntheticpeptidecorrespondi
5、ngtothe22carboxyl-terminalCOScellsandAtT-20cellsexpressingtheseproteinsfromaminoacidsofElandaffinitypurifiedasdescribed(5).recombinantvacciniavectors.Byimmunoperoxidaseandim-ImmunoperoxidaseLabeling.AtT-20cellswereplatedinmunogoldlabeling,thewild-typeElproteinwaslocalizedto35-mmdishes2daysprior
6、toinfection.CellsthatwereaboutoneortwocisternaelocatedononesideoftheGolgistackthat80%confluentwereinfectedwithrecombinantvacciniavi-couldbeidentifiedasthecissideinAtT-20cells.Incontrast,rusesatamultiplicityofinfectionof"20,withadsorptionofthemutantElproteinwasdetectedinallcisternaeacrossthe0.25
7、mlofserum-freemediumfor30minat37TC.Mediumstackaswellasattheplasmamembrane.WhentheElproteinscontainingserumwasthenadded,andcellswerefixedat4hrwereimmunoprecipitatedandsubjectedtodigestionwithafterinfectionin3%paraformaldehyde/0.05%
