Effect of foot-and-mouth disease virus capsid precursor protein and 3C protease expression on bovine herpesvirus 1 replication.

Several reports have previously shown that expression of the foot-and-mouth disease virus (FMDV) capsid precursor protein encoding region P1-2A together with the 3C protease (P1-2A/3C) results in correct processing of the capsid precursor into VP0, VP1 and VP3 and formation of FMDV capsid structures that are able to induce a protective immune response against FMDV challenge after immunization using naked DNA constructs or recombinant viruses. To elucidate whether bovine herpesvirus 1 (BHV-1) might also be suitable as a viral vector for empty capsid generation, we aimed to integrate a P1-2A/3C expression cassette into the BHV-1 genome, which, however, failed repeatedly. In contrast, BHV-1 recombinants that expressed an inactive 3C protease or the P1-2A polyprotein alone could be easily generated, although the recombinant that expressed P1-2A exhibited a defect in direct cell-cell spread and release of infectious particles.

Protein display by bovine herpesvirus type 1 glycoprotein B.

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.

Novel vectors for simultaneous high-level dual protein expression in vertebrate and insect cells by recombinant baculoviruses.

Gene transfer into cells of mammalian, avian or piscine origin by baculoviruses carrying expression cassettes active in vertebrate cells (BacMam method) is an attractive alternative to chemical or physical transfection methods or to the use of vectors originating from viruses of vertebrates. For simultaneous high-level expression of two proteins from recombinant baculoviruses we constructed novel dual expression vectors containing human and murine cytomegalovirus immediate-early enhancer/promoter elements in combination with the baculoviral polyhedrin and p10 promoters for simultaneous expression in vertebrate and insect cells. Transduction of ruminant cells with BacMam viruses containing the green fluorescent protein open reading frame downstream from the respective enhancer/promoter elements revealed that a dual expression cassette combining the murine cytomegalovirus immediate-early 1 sequence with the immediate early enhancer/promoter of human cytomegalovirus yields high levels of protein from both transcription units.