title: High-level expression of biologically active bovine alpha interferon by Bovine herpesvirus 1 interferes only marginally with recombinant virus replication in vitro.
abstract: An artificial open reading frame (ORF) for bovine alpha interferon (boIFN-alpha) with the codon preference of Bovine herpesvirus 1 (BHV-1) glycoprotein B was constructed to assess the effect of expression of boIFN-alpha by BHV-1 from an expression cassette. Transient expression of the ORF revealed that transfected cells secreted substantial amounts of biologically active boIFN-alpha, which moderately inhibited replication of BHV-1 after stimulation of bovine cells with 10(4) U ml(-1). The boIFN-alpha-encoding expression cassette was recombined into the glycoprotein E locus of the glycoprotein E-negative BHV-1 vaccine strain GKD. Cells infected with the resulting recombinant BHV-1/boIFN-alpha secreted up to 10(7) U boIFN-alpha per ml cell culture supernatant, which is about 40- to more than 100-fold the activity reached with other virus expression systems. Bioassays demonstrated that the BHV-1-expressed interferon induced a rapid and sustained antiviral state in stimulated bovine cells. Analysis of the in vitro growth properties of the recombinant revealed, depending on the cell line used, no or only slight inhibition in direct spreading from cell to cell and a modest delay in virus egress from infected cells. Final titres, however, were comparable to those reached by the parent strain. Penetration into cells was not affected. The results from this study demonstrate that BHV-1/boIFN-alpha expresses high levels of boIFN-alpha, grows to high titres in cell culture and thus represents a potential alternative means to deliver endogenously produced boIFN-alpha in situ for a period of time.
authors.affiliation: Institute of Molecular Biology, Friedrich-Loeffler-Institut, Boddenblick 5A, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Constanze
authors.initials: C
authors.lastname: Höhle
authors.affiliation: Institute of Molecular Biology, Friedrich-Loeffler-Institut, Boddenblick 5A, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Karger
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Boddenblick 5A, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Patricia
authors.initials: P
authors.lastname: König
authors.affiliation: Institute of Molecular Biology, Friedrich-Loeffler-Institut, Boddenblick 5A, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Giesow
authors.affiliation: Institute of Molecular Biology, Friedrich-Loeffler-Institut, Boddenblick 5A, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Günther M
authors.initials: GM
authors.lastname: Keil
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003062:Codon / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D007242:Herpesvirus 1, Bovine / Q000254:growth & development / Q000378:metabolism*; D016898:Interferon-alpha / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D003062:Codon; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C046998:bovine herpesvirus type-1 glycoproteins