1887

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Volume 85, Issue 12

Thermostability of the human respiratory syncytial virus fusion protein before and after activation: implications for the membrane-fusion mechanism Free

Abstract

Anchorless fusion (F) proteins () of human respiratory syncytial virus (RSV) are seen by electron microscopy as unaggregated cones when the proteolytic cleavage at two furin sites required for membrane-fusion activity is incomplete, but aggregate into rosettes of lollipop-shaped spikes following cleavage. To show that this aggregation occurred by interactions of the fusion peptide, a deletion mutant of lacking the first half of the fusion peptide was generated. This mutant remained unaggregated even after completion of cleavage, supporting the notion that aggregation of involved the fusion peptide. As exposure of the fusion peptide is a key event that occurs after activation of F proteins, the uncleaved and cleaved forms of may represent the pre- and post-active forms of RSV F protein. In an analysis of the structural differences between the two forms, their thermostability before and after proteolytic cleavage was examined. In contrast to other viral proteins involved in membrane fusion (e.g. influenza haemagglutinin), the pre-active (uncleaved) and post-active (cleaved) forms of were equally resistant to heat denaturation, assessed by spectrofluorimetry, circular dichroism or antibody binding. These results are interpreted in terms of the proposed structural changes associated with the process of membrane fusion mediated by RSV F protein.

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2004-12-01
2024-04-25
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References

  1. Bembridge G. P., Lopez J. A., Bustos R., Melero J. A., Cook R., Mason H., Taylor G. 1999; Priming with a secreted form of the fusion protein of respiratory syncytial virus (RSV) promotes interleukin-4 (IL-4) and IL-5 production but not pulmonary eosinophilia following RSV challenge. J Virol 73:10086–10094
     [Google Scholar]
  2. Blasco R., Moss B. 1995; Selection of recombinant vaccinia viruses on the basis of plaque formation. Gene 158:157–162 [CrossRef]
     [Google Scholar]
  3. 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]
  4. Bukreyev A., Whitehead S. S., Murphy B. R., Collins P. L. 1997; Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse. J Virol 71:8973–8982
     [Google Scholar]
  5. Calder L. J., González-Reyes L., García-Barreno B., Wharton S. A., Skehel J. J., Wiley D. C., Melero J. A. 2000; Electron microscopy of the human respiratory syncytial virus fusion protein and complexes that it forms with monoclonal antibodies. Virology 271:122–131 [CrossRef]
     [Google Scholar]
  6. Collins P. L., Mottet G. 1993; Membrane orientation and oligomerization of the small hydrophobic protein of human respiratory syncytial virus. J Gen Virol 74:1445–1450 [CrossRef]
     [Google Scholar]
  7. Collins P. L., Huang Y. T., Wertz G. W. 1984; Nucleotide sequence of the gene encoding the fusion (F) glycoprotein of human respiratory syncytial virus. Proc Natl Acad Sci U S A 81:7683–7687 [CrossRef]
     [Google Scholar]
  8. Collins P. L., Chanock R. M., Murphy B. R. 2001; Respiratory syncytial virus. In Fields Virology , 4th edn. pp  1443–1485 Edited by Knipe D. M., Howley P. M. Philadelphia, PA: Lippincott Williams & Wilkins;
     [Google Scholar]
  9. Durrer P., Galli C., Hoenke S., Corti C., Glück R., Vorherr T., Brunner J. 1996; H+-induced membrane insertion of influenza virus hemagglutinin involves the HA2 amino-terminal fusion peptide but not the coiled coil region. J Biol Chem 271:13417–13421 [CrossRef]
     [Google Scholar]
  10. Falsey A. R., Cunningham C. K., Barker W. H., Kouides R. W., Yuen J. B., Menegus M., Weiner L. B., Bonville C. A., Betts R. F. 1995; Respiratory syncytial virus and influenza A infections in the hospitalized elderly. J Infect Dis 172:389–394 [CrossRef]
     [Google Scholar]
  11. García-Barreno B., Jorcano J. L., Aukenbauer T., López-Galíndez C., Melero J. A. 1988; Participation of cytoskeletal intermediate filaments in the infectious cycle of human respiratory syncytial virus (RSV). Virus Res 9:307–321 [CrossRef]
     [Google Scholar]
  12. García-Barreno B., Palomo C., Peñas C., Delgado T., Perez-Breña P., Melero J. A. 1989; Marked differences in the antigenic structure of human respiratory syncytial virus F and G glycoproteins. J Virol 63:925–932
     [Google Scholar]
  13. García-Barreno B., Delgado T., Melero J. A. 1996; Identification of protein regions involved in the interaction of human respiratory syncytial virus phosphoprotein and nucleoprotein: significance for nucleocapsid assembly and formation of cytoplasmic inclusions. J Virol 70:801–808
     [Google Scholar]
  14. Glezen W. P., Taber L. H., Frank A. L., Kasel J. A. 1986; Risk of primary infection and reinfection with respiratory syncytial virus. Am J Dis Child 140:543–546
     [Google Scholar]
  15. González-Reyes L., Ruiz-Argüello M. B., García-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]
  16. Heminway B. R., Yu Y., Tanaka Y., Perrine K. G., Gustafson E., Bernstein J. M., Galinski M. S. 1994; Analysis of respiratory syncytial virus F, G, and SH proteins in cell fusion. Virology 200:801–805 [CrossRef]
     [Google Scholar]
  17. Karron R. A., Buonagurio D. A., Georgiu A. F. 8 other authors 1997; Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro : clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant. Proc Natl Acad Sci U S A 94:13961–13966 [CrossRef]
     [Google Scholar]
  18. Lamb R. A. 1993; Paramyxovirus fusion: a hypothesis for changes. Virology 197:1–11 [CrossRef]
     [Google Scholar]
  19. Lawless-Delmedico M. K., Sista P., Sen R. 7 other authors 2000; Heptad-repeat regions of respiratory syncytial virus F1 protein form a six-membered coiled-coil complex. Biochemistry 39:11684–11695 [CrossRef]
     [Google Scholar]
  20. Levine S., Klaiber-Franco R., Paradiso P. R. 1987; Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus. J Gen Virol 68:2521–2524 [CrossRef]
     [Google Scholar]
  21. López J. A., Bustos R., Örvell C., Berois M., Arbiza J., García-Barreno B., Melero J. A. 1998; Antigenic structure of human respiratory syncytial virus fusion glycoprotein. J Virol 72:6922–6928
     [Google Scholar]
  22. Matthews J. M., Young T. F., Tucker S. P., Mackay J. P. 2000; The core of the respiratory syncytial virus fusion protein is a trimeric coiled coil. J Virol 74:5911–5920 [CrossRef]
     [Google Scholar]
  23. Pastey M. K., Samal S. K. 1997; Analysis of bovine respiratory syncytial virus envelope glycoproteins in cell fusion. J Gen Virol 78:1885–1889
     [Google Scholar]
  24. Ruigrok R. W. H., Martín S. R., Wharton S. A., Skehel J. J., Bayley P. M., Wiley D. C. 1986; Conformational changes in the hemagglutinin of influenza virus which accompany heat-induced fusion of virus with liposomes. Virology 155:484–497 [CrossRef]
     [Google Scholar]
  25. Ruiz-Argüello M. B., González-Reyes L., Calder L. J., Palomo C., Martín D., Saíz M. J., García-Barreno B., Skehel J. J., Melero J. A. 2002; Effect of proteolytic processing at two distinct sites on shape and aggregation of an anchorless fusion protein of human respiratory syncytial virus and fate of the intervening segment. Virology 298:317–326 [CrossRef]
     [Google Scholar]
  26. Skehel J. J., Wiley D. C. 2000; Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69:531–569 [CrossRef]
     [Google Scholar]
  27. Srinivasakumar N., Ogra P. L., Flanagan T. D. 1991; Characteristics of fusion of respiratory syncytial virus with HEp-2 cells as measured by R18 fluorescence dequenching assay. J Virol 65:4063–4069
     [Google Scholar]
  28. Techaarpornkul S., Barretto N., Peeples M. E. 2001; Functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene. J Virol 75:6825–6834 [CrossRef]
     [Google Scholar]
  29. Walsh E. E., Hruska J. 1983; Monoclonal antibodies to respiratory syncytial virus proteins: identification of the fusion protein. J Virol 47:171–177
     [Google Scholar]
  30. Wertz G. W., Collins P. L., Huang Y., Gruber C., Levine S., Ball L. A. 1985; Nucleotide sequence of the G protein gene of human respiratory syncytial virus reveals an unusual type of viral membrane protein. Proc Natl Acad Sci U S A 82:4075–4079 [CrossRef]
     [Google Scholar]
  31. Wharton S. A., Skehel J. J., Wiley D. C. 2000; Temperature dependence of fusion by Sendai virus. Virology 271:71–78 [CrossRef]
     [Google Scholar]
  32. Wrigley N. G., Brown E. B., Skehel J. J. 1986; Electron microscopy of influenza virus. In Electron Microscopy of Proteins vol. 5 pp  103–163 Edited by Harris J. R., Horne R. W. London: Academic Press;
     [Google Scholar]
  33. Zhao X., Singh M., Malashkevich V. N., Kim P. S. 2000; Structural characterization of the human respiratory syncytial virus fusion protein core. Proc Natl Acad Sci U S A 97:14172–14177 [CrossRef]
     [Google Scholar]
  34. Zimmer G., Budz L., Herrler G. 2001; Proteolytic activation of respiratory syncytial virus fusion protein. Cleavage at two furin consensus sequences. J Biol Chem 276:31642–31650 [CrossRef]
     [Google Scholar]
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Thermostability of the human respiratory syncytial virus fusion protein before and after activation: implications for the membrane-fusion mechanism
J. Gen. Virol. 85, 3677 (2004); https://doi.org/10.1099/vir.0.80318-0
/content/journal/jgv/10.1099/vir.0.80318-0
/content/journal/jgv/10.1099/vir.0.80318-0
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