title: Recombinant bovine herpesvirus-1 (BHV-1) lacking transactivator protein BICPO entails lack of glycoprotein C and severely reduced infectivity.
abstract: The immediate-early transactivator protein BICPO is a key regulatory element of bovine herpesvirus 1 (BHV-1) replication based on transient expression assays. To examine BICPO function in the context of the viral genome, we created recombinant BHV-1 expressing beta-galactosidase instead of BICPO. To complement the defect, a neomycin resistant MDBK cell line (M164) expressing BICPO was established, permitting selection of a blue-staining BHV-1 recombinant (A2G2). Southern blot and PCR analysis confirmed that the BICPO gene was interrupted by the beta-galactosidase gene and that wt progeny was absent. Compared with wt BHV-1, A2G2 reached lower titers in M164 cells but replicated with similar kinetics. Once isolated, A2G2 also grew in MDBK cells although the titer was reduced a further 10-fold and the virus remained strongly cell-associated. Thus, BICPO is not absolutely required for replication in cell culture. Gene expression of A2G2 was investigated by Western blots and immunofluorescence. Surprisingly, not only was BICPO absent, but glycoprotein C (gC) was also missing. Other viral genes were expressed normally. Semiquantitative PCR showed that A2G2 produced similar amounts of viral DNA as wt but a much smaller number of infectious particles. Cotransfection of A2G2 DNA and a plasmid containing the BICPO gene yielded revertant virus with fully restored wt properties. We conclude that BICPO is required for gC expression, and that the missing gC partly accounts for the reduced A2G2 infectivity.
authors.affiliation: Institute of Virology, Faculty of Veterinary Medicine, University of Zürich, Switzerland.
authors.forename: R
authors.initials: R
authors.lastname: Köppel
authors.forename: C
authors.initials: C
authors.lastname: Fraefel
authors.forename: B
authors.initials: B
authors.lastname: Vogt
authors.forename: L J
authors.initials: LJ
authors.lastname: Bello
authors.forename: W C
authors.initials: WC
authors.lastname: Lawrence
authors.forename: M
authors.initials: M
authors.lastname: Schwyzer
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D004285:Dogs; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D018139:Simplexvirus / Q000235:genetics / Q000472:pathogenicity*; D015534:Trans-Activators / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D014759:Viral Envelope Proteins / Q000172:deficiency*; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D011994:Recombinant Proteins; D015534:Trans-Activators; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C038535:glycoprotein gC, herpes simplex virus type 1; D044767:Ubiquitin-Protein Ligases; C086729:bICP0 protein, Bovine herpesvirus 1