title: Protein display by bovine herpesvirus type 1 glycoprotein B.
abstract: Glycoprotein B (gB) of bovine herpesvirus 1 (BHV-1), a major component of the viral envelope, is essential for membrane fusion during entry and cell-to-cell spread. It is cleaved in the trans-Golgi network by the proprotein convertase furin. Integration of the open reading frame (ORF) encoding a mutated gB with a second furin cleavage site and mature boIFN-alpha as intervening peptide between the amino-terminal (NH(2)) and carboxy-terminal (COOH) gB subunits yielded recombinant BHV-1/gB2FuIFN-alpha which, unexpectedly, express gB with an enlarged NH(2)-subunit of 90kDa. Here we show that boIFN-alpha-specific antibodies bind to the 90kDa gB subunit and efficiently neutralize BHV-1/gB2FuIN-alpha infectivity. We also show that inactivated BHV-1/gB2FuIN-alpha virions induce an antiviral state in cells incubated with UV-inactivated particles. These results demonstrate that the 90kDa protein is a NH(2)-subunit/boIFN-alpha fusion protein whose boIFN-alpha domain is biologically active. To verify that BHV-1 gB is suitable for the display of (glyco)proteins on the surface of virions we constructed BHV-1 recombinants expressing within gB the first 273 amino acids of the NH(2)-subunit (HA1) of avian influenza haemagglutinin, either flanked by two furin cleavage sites or with only one cleavage site between a gB/NH(2)_HA1 fusion protein and the COOH subunit. The resulting recombinant BHV-1/gB2FuHA1 expressed gB from which 55kDa HA1 was excised and secreted. In contrast, gB from BHV-1/gB_NH(2)HA1 infected cells retained HA1 as fusion protein with the NH(2)-subunit. Immunoblotting and neutralization analyses revealed that HA1 is incorporated into the envelope BHV-1/gB/NH(2)_HA1 particles and exposed to the exterior of virions. Thus, this novel approach enables display of polypeptides and (glyco)proteins of at least 273 amino acids on viral particles which is of particular interest for development of novel diagnostics and vaccines as well as for, e.g. gene therapy applications especially when biologically active ligands need to be presented.
authors.affiliation: Friedrich-Loeffler-Institut, Boddenblick 5A, 17493 Greifswald-Insel Riems, Germany. Guenther.Keil@fli.bund.de
authors.forename: Günther M
authors.initials: GM
authors.lastname: Keil
authors.forename: Constanze
authors.initials: C
authors.lastname: Klopfleisch
authors.forename: Katrin
authors.initials: K
authors.lastname: Giesow
authors.forename: Jutta
authors.initials: J
authors.lastname: Veits
mesh_terms: D000818:Animals; D002417:Cattle; D045683:Furin / Q000378:metabolism; D005821:Genetic Techniques; D007242:Herpesvirus 1, Bovine / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D016366:Open Reading Frames; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C046998:bovine herpesvirus type-1 glycoproteins; D045683:Furin