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1、VIROLOGY142,378-388(1985)AttenuationofMurineCoronavirusInfectionbyAmmoniumChlorideLEEMIZZEN,’ANNEHILTON,STEVECHELEY,”ANDROBERTANDERSON’p3DepartmentofMicrobiologyandImmunology,UniversityofWesternOntario,Londm,OntarioN6ASCl,CanadaReceivedAugust13,1984acceptedDecember4,1984Ammoniumchl
2、orideataconcentrationof20mMdelayedby4-5hrtheproductionofvirusprogenyinmouseL-2cellsinfectedathighmultiplicitywithmousehepatitisvirus(MHV).Thisdelaywasseenintheproductionofbothintracellularandextracellularvirus.However,thefinaltitersweresimilartothoseproducedbyMHV-infectedcellsmaint
3、ainedinnormalmedium.Themanifestationofvirus-inducedcellfusionwassimilarlyfoundtobedelayed,butnototherwisedecreasedinseverity,whenammoniumchloridewaspresentintheculturemedium.Ammoniumchloridecausedsimilardelaysinproductionofvirus-specific,positive-senseRNAsandofviralpolypeptides.The
4、relativeproportionsandapparentmolecularweightsofviralRNAsandpolypeptidesweresimilartothosefoundinMHV-infectedcellsculturedinnormalmedium.InvitrotranslationofendogenouslyproducedviralRNAsincellextracts,preparedfromMHV-infectedcells,wasnotinhibitedbyammoniumchloride.Thus,ammoniumchlo
5、ridehasnospecific,inhibitoryeffectonviralproteinsynthesis.Ammoniumchloridedidnotreducethenumberofvirus-infectedcellsinculture,asmonitoredbyinfectiouscenterassay.AnalysisofearlyeventsinMHVinfectionshowedthatammoniumchloridedidnotaffectadsorptionorinternalizationofMHVbyL-2cells.Howev
6、er,thesubsequenteclipsephase,asmonitoredbydeclineininfectivityofinternalizedvirusinoculumproceededlessefficientlyinthepresenceofammoniumchloride.Onthebasisoftheknowninhibitoryeffectsofammoniumchlorideonlysosomal/endosomalfunctions,theresultssuggestanendosomalmechanismofMHVuncoating
7、.ThustheprimaryeffectofammoniumchlorideonMHVinfectionofL-2cellsistoattenuatevirusuncoating,therebychronologicallydisplacingallsubsequentvirus-encodedfunctions.o1985AcademicPress,Inc.INTRODUCTIONendocyticmechanism(Dales,1973;FanandSefton,1978;MarshandHelenius,Theearliesteventsinvolv
8、edinvirus1980;Simpsonetcd,