title: Equine herpesvirus type 1 devoid of gM and gp2 is severely impaired in virus egress but not direct cell-to-cell spread.
abstract: Experiments were conducted to analyze the effects of a simultaneous deletion of glycoprotein M (gM) and glycoprotein 2 (gp2) of equine herpesvirus type 1 (EHV-1). EHV-1 strain RacH was cloned as a bacterial artificial chromosome (pRacH) by homologous recombination of a mini F plasmid into the unique short region of the genome, thereby deleting gene 71 encoding gp2. Upon transfection of the pRacH DNA into rabbit kidney RK13 cells, virus plaques were visible from day 1 after transfection. The mutant RacH virus (H Delta gp2) reconstituted from pRacH lacked gene 71 and did not express gp2 as assayed by indirect immunofluorescence analysis using gp2-specific monoclonal antibodies. The H Delta gp2 virus exhibited 10-fold reduced extracellular titers and an approximately 10% reduction in mean plaque diameters when compared to parental or gp2-revertant virus. The gM open reading frame was deleted from pRacH by recE/T mediated mutagenesis in Escherichia coli. The gM-gp2 double negative virus mutant (H Delta gp2gM) did not express either of the deleted glycoproteins as demonstrated by indirect immunofluorescence analysis. The H Delta gp2gM virus exhibited a 200-fold reduction of end-point extracellular titers when compared to parental RacH virus, which could not be compensated for by growth of the mutant virus on gM-expressing cells. After restoration of the gM open reading frame, however, growth of the mutant virus was comparable to the H Delta gp2 virus. Plaque diameters of the gM-gp2 double-negative mutant were reduced by only 16% when compared to that of parental RacH virus. From the results it was concluded that the simultaneous absence of gM and gp2 had an additive effect on egress but not secondary envelopment or cell-to-cell spread of EHV-1.
authors.affiliation: Institute of Molecular Biology, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Rudolph
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Osterrieder
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D004861:Herpesvirus 1, Equid / Q000502:physiology*; D009154:Mutation; D016366:Open Reading Frames; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication
chemical_list: D014759:Viral Envelope Proteins; C418522:glycoprotein M, Equid herpesvirus 1; C077966:glycoprotein 300, Equid herpesvirus 1