title: Molecular characterization and baculovirus expression of the glycoprotein B of a seal herpesvirus (phocid herpesvirus-1).
abstract: A glycoprotein B (gB) gene homologue was identified in a 5.4-kb BamHl genomic fragment of the phocid herpesvirus type-1 (PhHV-1) which represents a widespread and important pathogen of pinnipeds. Sequence analysis revealed a gB-specific open-reading frame comprising 881 amino acids. Phylogenetic analysis gave evidence for a close evolutionary relationship between PhHV-1 and members of the Varicellovirus genus of the alpha-Herpesvirinae and canid herpesvirus in particular. In PhHV-1-infected Crandell feline kidney cells gB is expressed as a 113-kDa glycosylated molecule which is proteolytically cleaved into at least two fragments of 67 and 53-59 kDa apparently forming disulfide-linked heterodimers of 140 kDa. Cell surface expression of PhHV-1 gB was confirmed by FACS analysis. Thus, synthesis and processing of the gB protein of PhHV-1 follows a pattern also observed in other Varicelloviruses. Since the gB protein of herpesviruses, expressed in the baculovirus system, has been shown to be a suitable target for vaccine design, we used this system for expression of PhHV-1 gB. Recombinant (rec) baculovirus-expressed gB was identified as a 105-kDa glycosylated molecule. Proteolytic cleavage into fragments of 62 and 52 kDa was markedly delayed compared to wild-type (wt) gB. Wt and rec gB harbored endoglycosidase H (precursor)- as well as N-glycosidase F-sensitive N-glycans (proteolytic fragments). Baculovirus-expressed gB appeared to be antigenically authentic, since it was recognized in radioimmunoprecipitation and immune peroxidase monolayer assays by PhHV-1-neutralizing seal sera and by gB-specific neutralizing murine monoclonal antibodies. Furthermore, PhHV-1-neutralizing antibodies were induced in mice following immunization with baculovirus-expressed gB, indicating its suitability for incorporation in a candidate vaccine for seals.
authors.affiliation: Department of Virology, Erasmus University Rotterdam, The Netherlands.
authors.forename: T C
authors.initials: TC
authors.lastname: Harder
authors.forename: A D
authors.initials: AD
authors.lastname: Osterhaus
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D002415:Cats; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D005814:Genes, Viral; D007668:Kidney; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology; D015183:Restriction Mapping; D046048:Seals, Earless; D018411:Spodoptera; D014162:Transfection; D018140:Varicellovirus / Q000235:genetics / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics
chemical_list: D004279:DNA, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins; C104210:glycoprotein B, phocid herpesvirus 1