High-level expression of biologically active bovine alpha interferon by Bovine herpesvirus 1 interferes only marginally with recombinant virus replication in vitro.

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.

Engineering glycoprotein B of bovine herpesvirus 1 to function as transporter for secreted proteins: a new protein expression approach.

Glycoprotein B (gB) of bovine herpesvirus 1 (BHV-1) is essential for BHV-1 replication and is required for membrane fusion processes leading to virus penetration into the target cell and direct spreading of BHV-1 from infected to adjacent noninfected cells. Like many of the herpesvirus gB homologs, BHV-1 gB is proteolytically processed by furin, an endoproteinase localized in the trans-Golgi network. Cleavage by furin is a common mechanism for the activation of a number of viral fusion (F) proteins. Among these, the F proteins of both human and bovine respiratory syncytial virus (RSV) have the so-far unique feature that cleavage of the respective F protein precursors occurs at two furin recognition sites, resulting in the release of a 27-amino-acid intervening peptide which is secreted into the extracellular space. We showed recently that the intervening peptide of bovine RSV can be replaced by bovine interleukins which are secreted into the medium of cells infected with the respective bovine RSV recombinants (P. Konig, K. Giesow, K. Schuldt, U. J. Buchholz, and G. M. Keil, J. Gen. Virol. 85:1815-1824, 2004). To elucidate whether the approach to transport heterologous proteins as furin-excisable polypeptides functions in principle also in glycoproteins which are cleaved by furin only once, we inserted a second furin cleavage site into BHV-1 gB and integrated a 16-amino-acid peptide sequence, the 246-amino-acid green fluorescent protein (GFP), or the 167 amino acids for mature bovine alpha interferon (boIFN-alpha) as an intervening polypeptide. The resulting gB variants rescued gB-negative BHV-1 mutants, the resulting BHV-1 recombinants were fully infectious, and infected cells secreted biologically active GFP and boIFN-alpha, respectively. In contrast to the gB2Fu and gB2FuGFP precursor molecules, which were efficiently cleaved at both furin sites, the majority of pgB2FuIFN-alpha was not cleaved at the site between the amino-terminal (NH2) subunit and boIFN-alpha, whereas cleavage at the newly introduced site was normal. This resulted in virus particles that also contain the NH2-subunit/boIFN-alpha fusion protein within their envelopes. Our results demonstrate that BHV-1 gB can be used as a transporter for peptides and proteins which could be important for development of novel vaccines. In addition, the general principle might be useful for other applications, e.g., in gene therapy and also in nonviral systems.