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A novel protein expression strategy using recombinant bovine respiratory syncytial virus (BRSV): modifications of the peptide sequence between the two furin cleavage sites of the BRSV fusion protein yield secreted proteins, but affect processing and function of the BRSV fusion protein

Ursula J. Buchholz

Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, 17493 Greifswald-Insel Riems, Germany

Correspondence
Günther M. Keil
Guenther.M.Keil{at}rie.bfav.de

The bovine respiratory syncytial virus (BRSV) fusion (F) protein is cleaved at two furin cleavage sites, which results in generation of the disulfide-linked F₁ and F₂ subunits and release of an intervening peptide of 27 aa (pep27). A series of mutated open reading frames encoding F proteins that lacked the entire pep27, that contained an arbitrarily chosen 23 aa sequence instead of pep27 or in which pep27 was replaced by the amino acid sequences for the bovine cytokines interleukin 2 (boIL2), interleukin 4 (boIL4) or gamma interferon (boIFN-) was constructed. Transient expression experiments revealed that the sequence of the intervening peptide influenced intracellular transport, maturation of the F protein and F-mediated syncytium formation. Expression of boIL2, boIL4 or boIFN- in place of pep27 resulted in secretion of the cytokines into the culture medium. All mutated F proteins except the boIFN--containing variant could be expressed by and were functional for recombinant BRSV. Characterization of the cell culture properties of the recombinants demonstrated that the amino acid sequence between the two furin cleavage sites affected entry into target cells, direct spreading of virions from cell to cell and virus growth. Secretion of boIL2 and boIL4 into the medium of cells infected with the respective recombinants demonstrated that the F protein can be used to express secreted heterologous bioactive peptides or (glyco)proteins, which might be of interest for the development of novel RSV vaccines.

Present address: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-8007, USA.

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