title: Cytomegalovirus assemblin (pUL80a): cleavage at internal site not essential for virus growth; proteinase absent from virions.
abstract: The human cytomegalovirus (HCMV) maturational proteinase is synthesized as an enzymatically active 74-kDa precursor that cleaves itself at four sites. Two of these, called the maturational (M) and release (R) sites, are conserved in the homologs of all herpesviruses. The other two, called the internal (I) and cryptic (C) sites, have recognized consensus sequences only among cytomegalovirus (CMV) homologs and are located in the 28-kDa proteolytic portion of the precursor, called assemblin. I-site cleavage cuts assemblin in half without detected effect on its enzymatic behavior in vitro. To investigate the requirement for this cleavage during virus infection, we used the CMV-bacterial artificial chromosome system (E. M. Borst, G. Hahn, U. H. Koszinowski, and M. Messerle, J. Virol. 73:8320-8329, 1999) to construct a virus encoding a mutant I site (Ala143 to Val) intended to be blocked for cleavage. Characterizations of the resulting mutant (i) confirmed the presence of the mutation in the viral genome and the inability of the mutant virus to effect I-site cleavage in infected cells; (ii) determined that the mutation has no gross effect on the rate of virus production or on the amounts of extracellular virions, noninfectious enveloped particles (NIEPs), and dense bodies; (iii) established that assemblin and its cleavage products are present in NIEPs but are absent from CMV virions, an apparent difference from what is found for virions of herpes simplex virus; and (iv) showed that the 23-kDa protein product of C-site cleavage is more abundant in mutant virus-than in wild-type virus-infected cells and NIEPs. We conclude that the production of infectious CMV requires neither I-site cleavage of assemblin nor the presence of assemblin in the mature virion.
authors.affiliation: Virology Laboratories, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Chee-Kai
authors.initials: CK
authors.lastname: Chan
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Brignole
authors.forename: Wade
authors.initials: W
authors.lastname: Gibson
mesh_terms: D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D003587:Cytomegalovirus / Q000201:enzymology* / Q000254:growth & development* / Q000472:pathogenicity; D003588:Cytopathogenic Effect, Viral; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D014771:Virion / Q000201:enzymology* / Q000378:metabolism; D019065:Virus Assembly
chemical_list: D010450:Endopeptidases; D012697:Serine Endopeptidases; C071910:assemblin