1887

Abstract

The bovine respiratory syncytial virus (BRSV) fusion (F) protein is cleaved at two furin cleavage sites, which results in generation of the disulfide-linked F and F 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.

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2004-07-01
2024-03-29
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References

  1. Buchholz U. J., Finke S., Conzelmann K.-K. 1999; Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture and the human RSV leader region acts as a functional BRSV genome promoter. J Virol 73:251–259
    [Google Scholar]
  2. Buchholz U. J., Granzow H., Schuldt K., Whitehead R. R., Murphy B. R., Collins P. L. 2000; Chimeric bovine respiratory syncytial virus with glycoprotein gene substitution from human respiratory syncytial virus (HRSV): effects on host range and evaluation as a live-attenuated HRSV vaccine. J Virol 74:1187–1199 [CrossRef]
    [Google Scholar]
  3. Bühler B., Keil G. M., Weiland F., Koszinowski U. H. 1990; Characterization of the murine cytomegalovirus early transcription unit e1 that is induced by immediate-early proteins. J Virol 64:1907–1919
    [Google Scholar]
  4. Bukreyev A., Whitehead S. S., Bukreyeva N., Murphy B. R., Collins P. L. 1999; Interferon gamma expressed by recombinant respiratory syncytial virus attenuates virus replication in mice without compromising immunogenicity. Proc Natl Acad Sci U S A 96:2367–2372 [CrossRef]
    [Google Scholar]
  5. Collins P. L., McIntosh K., Chanock R. M. 2001; Respiratory syncytial virus. In Fields Virology , 4th edn. pp  1443–1485 Edited by Knipe D. M., Howley P. M., Griffin D. E., Lamb R. A., Martin A., Roizman B., Straus S. E. Philadelphia: Lippincot, Williams & Wilkins;
    [Google Scholar]
  6. Gonzáles-Reyes L., Ruiz-Argüello M. B., Garcia-Barreno B., Calder L., López J. A., Albar J. P., Skehel J. J., Wiley D. C., Melero J. A. 2001; Cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion. Proc Natl Acad Sci U S A 98:9859–9864 [CrossRef]
    [Google Scholar]
  7. Karger A., Schmidt U., Buchholz U. J. 2001; Recombinant bovine respiratory syncytial virus with deletions of the G or SH genes: G and F proteins bind heparin. J Gen Virol 82:631–640
    [Google Scholar]
  8. Keil G. M., Fibi M. R., Koszinowski U. H. 1985; Characterization of the major immediate-early polypeptides encoded by murine cytomegalovirus. J Virol 54:422–428
    [Google Scholar]
  9. Klenk H. D., Garten W. 1994; Host cell proteases controlling viral pathogenicity. Trends Microbiol 2:39–43 [CrossRef]
    [Google Scholar]
  10. Koszinowski U. H., Keil G. M., Volkmer H., Fibi M. R., Ebeling-Keil A., Münch K. 1986; The 89,000-Mr murine cytomegalovirus immediate-early protein activates gene transcription. J Virol 58:59–66
    [Google Scholar]
  11. Kühnle G., Collins R. A., Scott J. E., Keil G. M. 1996; Bovine interleukins 2 and 4 expressed in recombinant bovine herpesvirus 1 are biologically active secreted glycoproteins. J Gen Virol 77:2231–2240 [CrossRef]
    [Google Scholar]
  12. Lerch R. A., Stott E. J., Wertz G. W. 1989; Characterization of bovine respiratory syncytial virus proteins and mRNAs and generation of cDNA clones to viral mRNAs. J Virol 63:833–840
    [Google Scholar]
  13. Mallipeddi S. K., Samal S. K., Mohanty S. B. 1990; Analysis of polypeptides synthesized in bovine respiratory virus-infected cells. Arch Virol 115:23–36 [CrossRef]
    [Google Scholar]
  14. Pringle C. R. 1996; Virus taxonomy 1996 – a bulletin from the Xth International Congress of Virology in Jerusalem. Arch Virol 141:2251–2256 [CrossRef]
    [Google Scholar]
  15. Sambrook F., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  16. Zimmer G., Budz L., Herrler G. 2001; Proteolytic activation of respiratory syncytial virus fusion protein. J Biol Chem 276:31642–31650 [CrossRef]
    [Google Scholar]
  17. Zimmer G., Conzelmann K.-K., Herrler G. 2002; Cleavage of the furin consensus sequence RAR/KR109 and presence of the intervening peptide of the respiratory syncytial virus fusion protein are dispensable for virus replication in cell culture. J Virol 76:9218–9224 [CrossRef]
    [Google Scholar]
  18. Zimmer G., Rohn M., McGregor G. P., Schemann M., Conzelmann K.-K., Herrler G. 2003; Virokinin, a bioactive peptide of the tachykinin family, is released from the fusion protein of bovine respiratory syncytial virus. J Biol Chem 278:46854–46861 [CrossRef]
    [Google Scholar]
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