An Avirulent Mutant of Rabies Virus Is Unable To I

JOURNALOFVIROLOGY,0022-538X/98/$04.0010Jan.1998,p.273–278Vol.72,No.1Copyright©1998,AmericanSocietyforMicrobiologyAnAvirulentMutantofRabiesVirusIsUnableToInfectMotoneuronsInVivoandInVitroPATRICECOULON,1,2JEAN-PIERRETERNAUX,2ANNEFLAMAND,1ANDCHRISTINETUFFEREAU1*LaboratoiredeGe´ne´tiquedesVirus,CentreNationaldelaRechercheScientifique,91198GifsurYvetteCedex,1andUnite´deNeurocyberne´tiqueCellulaire,CentreNationaldelaRechercheScientifique,13009Marseille,2FranceReceived21July1997/Accepted23September1997Anantigenicdoublemutantofrabiesvirus(challengevirusstandard[CVS]strain)wasselectedbysucces-siveuseoftwoneutralizingantiglycoproteinmonoclonalantibodies,bothspecificforantigenicsiteIII.Thismutantdifferedfromtheoriginalvirusstrainbytwoaminoacidsubstitutionsintheectodomainoftheglyco-protein.Thelysineinposition330andthearginineinposition333werereplacedbyasparagineandmethi-onine,respectively.Thisdoublemutantwasnotpathogenicforadultmice.Wheninjectedintramuscularlyintotheforelimbsofadultmice,thisviruscouldnotpenetratethenervoussystem,eitherbythemotororbythesensoryroute,whilerespectivesinglemutantsinfectedmotoneuronsinthespinalcordandsensoryneuronsinthedorsalrootganglia.InvitroexperimentsshowedthatthedoublemutantwasabletoinfectBHKcells,neuroblastomacells,andfreshlypreparedembryonicmotoneurons,albeitwithalowerefficiencythantheCVSstrain.Uponfurtherincubationat37°C,themotoneuronsbecameresistanttoinfectionbythemutantwhileremainingpermissivetoCVSinfection.Theseresultssuggestthatrabiesvirususesdifferenttypesofreceptors:amoleculewhichisubiquitouslyexpressedatthesurfaceofcontinuouscelllinesandwhichisrecognizedbybothCVSandthedoublemutantandaneuron-specificmoleculewhichisnotrecognizedbythedoublemutant.Tissuetropismofavirusisfirstdeterminedbytheinterac-tionofviralsurfaceprotein(s)withmoleculesexpressedatthesurfaceoftargetcells.Expressionofsuchmoleculesinalim-itedgroupofdifferentiatedtissuesrestrictsthetropismofavirus.Rabiesvirusisaclearexampleofsuchasituation.Thisenvelopedvirus,whosegenomeisanonsegmentednegative-strandRNA,belongstotherhabdovirusfamily.Thegenomecodesforfiveproteins,oneofwhich,glycoprotein(G),isexposedatthesurfaceofthevirion(29).Invitro,thevirusisabletoinfectvarioustypesofcells(19).Invivoitstropismismostlyrestrictedtoneurons.However,afterintramuscularinoculation,rabiesviruscansimultaneouslyinfectneuronsandmusclecells(15).Replicationinmusclecells,whichisobservedparticularlywithstreetrabiesviruses,isnotrequiredforinfectionofthener-voussystem(NS)(6).DuringthecourseoftheNSinvasion,onlyneuronscontainviralantigens.Thisspecificityofneuronalinfectionledustopostulatetheexistenceofspecificreceptorsforrabiesvirusatthesurfaceofneurons.IndirectevidenceofspecificinteractionsbetweenGandneuronalmoleculeshasbeenfoundbytheuseofantigenicmutants.SelectionofvirusesmutatedintheGgeneispossiblebytheisolationofmutantsresistanttoneutralizationbymono-clonalantibodies(MAbs)fromamongasensitivepopulation.OnthebasisoftheirreactivitytowardacollectionofMAbs,MAb-resistant(MAR)mutantshavebeenclassifiedintodif-ferentgroupswhichdefineantigenicsites(4).Systematicin-oculationofmicewithMARmutantsshowedthatfewofthemexhibitedadrasticmodificationofpathogenicity.Allofthesemutantshadasubstitutionoftheirarginineatposition333ofG(21).ThismutationaffectedantigenicsiteIIIandgeneratedvirusesavirulentforallimmunocompetentadultanimalstested.Theonlyexceptionconcernedskunks,whichhavebeenreportedtobesusceptibletoinfectionbythistypeofmutant(25),buttheseresultshavenotbeenconfirmed.Tounderstandthereasonsforthislackofpathogenicity,oneoftheseavirulentmutants(AvO1),selectedfromthechallengevirusstandard(CVS)strain,hasbeenstudiedindetail.AllresultsobtainedfrommicesupporttheideathatAvO1isabletopenetratetheNSbutcaninfectonlyasubsetofneurons(7).Followingintramuscularinjection,AvO1asCVSinfectsmotorandsen-soryneuronswiththesameefficiency.However,unlikeCVS,themutantdoesnotinfectotherneuronsatlatertimesofinfection,eitherinthespinalcordorinthebrain(6).Afterintranasalinoculation,AvO1andCVSinfectfirstneuronsoftheolfactoryepithelium.Fromthere,AvO1istransmittedtoafewcategoriesofneuronsconnectedtoolfactoryreceptorcells(i.e.,periglomerularneuronsintheolfactorybulbandneuronsofthehorizontaldiagonalbandinthebrain),whileCVSin-vadesmostcategoriesofneuronsoftheolfactorysystem(mi-tralcells,neuronsoftheinternalplexiformlayerintheolfac-torybulbandanteriorolfactorynucleus,andperiglomerularcellsandneuronsofthehorizontaldiagonalband)(12).InthecaseofAvO1,restrictionofviralpropagationleavestimefortheimmunesystemtodevelopaspecificresponsewhichleadstoeliminationofthevirusfromthecentralNS(CNS).TheseresultsindicatethatAvO1maturesfromolfactoryreceptorcellsandsuggestthatthemutationpreventspenetrationinasubsetofconnectedneurons(12).AntigenicsiteIIIhasbeendescribedascoveringaminoacids330to338(21).Assumingthatotheraminoacidslocatedclosetoposition333arealsoimplicatedintheabilityofthevirustoinfectneurons,weselectedMARmutantswithtwomutationsinsiteIIIandstudiedtheirresidualneuroinvasiveness.Thispaperdescribestheselectionandmolecularcharacte