Molecular characterization and baculovirus expression of the glycoprotein B of a seal herpesvirus (phocid herpesvirus-1).
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.
|Keywords||0 (DNA, Viral), 0 (Recombinant Proteins), 0 (Viral Envelope Proteins), 0 (glycoprotein B, phocid herpesvirus 1), Amino Acid Sequence, Animals, Baculoviridae, Base Sequence, Cats, Cell Line, Cloning, Molecular, DNA, Viral/chemistry/metabolism, Genes, Structural, Viral, Kidney, Mice, Molecular Sequence Data, Open Reading Frames, Phylogeny, Polymerase Chain Reaction, Recombinant Proteins/biosynthesis/chemistry/immunology, Restriction Mapping, Seals, Spodoptera, Support, Non-U.S. Gov't, Transfection, Varicellovirus/genetics/*physiology, Viral Envelope Proteins/*biosynthesis/chemistry/genetics|
|Persistent URL||dx.doi.org/10.1006/viro.1996.8332, hdl.handle.net/1765/3591|
Harder, T.C., & Osterhaus, A.D.M.E.. (1997). Molecular characterization and baculovirus expression of the glycoprotein B of a seal herpesvirus (phocid herpesvirus-1).. Virology, 227(2), 343–352. doi:10.1006/viro.1996.8332