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Unformatted text preview: JOURNAL OF VIROLOGY, Mar. 1994,p.1532-1543 Vol.68,No. 3 0022-538X/94/$04.00+0 Copyright 1994,American SocietyforMicrobiology TranscriptionInhibitionand Other PropertiesofMatrixProteins Expressedby M Genes Cloned from Measles Viruses and DiseasedHuman BrainTissue KALACHAR SURYANARAYANA,' KNUT BACZKO,2VOLKER TER MEULEN,2 AND ROBERT R. WAGNER'* DepartmentofMicrobiologyand CancerCenter, UniversityofVirginiaMedical School, Charlottesville, Virginia 22908,1 andInstitut fur Virologie und Immunobiologie, UniversitdtWurzburg, 97078 Wiirzburg, Germany2 Received 13August 1993/Accepted 22November 1993 Ribonucleoprotein (RNP) coresextractedfromvirionsofwild-type(Edmonstonstrain)measlesvirus (MV) or obtained from MV-infected cells (cRNP) were shown to be capable oftranscribing RNA in vitrobut at relativelylowefficiency.The tightlybound matrix (M) proteincould be effectivelyremoved from virion RNP (vRNP) andfromcRNP byexposuretobuffersofhighionicstrength(0.5 to1.0M KCI) but onlyatpH 8.0or higher. The vRNP and cRNP cores complexed with M protein exhibited markedly reduced transcriptional activityatincreasingconcentrations,whereas vRNP and cRNP cores freeof M proteinexhibited linearand substantiallyhighertranscriptionalactivity;thesedata suggestthatM proteinistheendogenous inhibitorof MV RNP transcription. M-gene cDNA clones derivedfrom three strainsofwild-type (wt)MV and 10 clones frommRNAs isolatedfromthebraintissueofpatientswho had diedfromsubacutesclerosingpanencephalitis (SSPE) and from measlesinclusionbodyencephalitis(MIBE) were recloned inthe pTM-1 expressionvector drivenbythebacteriophageT7 RNA polymeraseexpressedbyacoinfectingvacciniavirusrecombinant. All10 mutant SSPE and MIBE clones expressed in vitro and in vivo M proteins that reacted with monospecific anti-M polyclonal antibody and migrated on polyacrylamide gels to positions identical to or only slightly differentfrom those ofthe M proteinsexpressed bywt MV clones.When reconstitutedwith cRNP cores, the three expressed wt M proteins and 6 ofthe 10 mutant-expressed M proteins showed equivalentcapacityto down-regulate MV transcription.Three ofthe M proteinsfrom SSPE clones and one from the MIBE clone showed littleor no capacity to down-regulate transcription when reconstituted with cRNP cores. The only plausibleexplanations forloss oftranscription inhibitionactivitybythefourSSPE/MIBE M proteinswere exceedinglyhighdegreesofhypermutations leadingtoU->C transitions and cloning-corrected mutations in the initiatorcodon (ATG->ACG) ofthefourM genes.However, onlythehypermutated M proteinexpressed bythe MIBE cDNA cloneexhibitedvirtuallynocapacitytobindcRNP cores ina reconstitutionassay.These experiments providesome preliminarydata tosupport thehypothesisthat MV encephalitis may resultfrom certain selectivemutations inthe M gene....
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