1887

Abstract

We have reported previously the expression and purification of an anchorless form of the human respiratory syncytial virus (HRSV) F protein () representing the ectodomain of the full-length F. molecules are seen as unaggregated cones by electron microscopy but completion of proteolytic cleavage of the F0 monomers in the trimer leads to a change in shape from cones to lollipops that aggregate into rosettes. This aggregation apparently occurs by interaction of the fusion peptides of molecules that are exposed after cleavage. Since exposure of the fusion peptide is a key event in the process of membrane fusion, changes associated with cleavage may reflect those occurring in full-length F during membrane fusion. Deletions or substitutions that changed either the length, charge or hydrophobicity of the fusion peptide inhibited aggregation of , and these mutants remained as unaggregated cones after cleavage. In contrast, more conservative changes did not inhibit the change of shape and aggregation of . When the same changes were introduced in the fusion peptide of full-length F, only the mutations that inhibited aggregation of prevented membrane fusion. Thus, the conformational changes that follow completion of cleavage of the protein require a functional fusion peptide. These sequence constraints may restrict accumulation of sequence changes in the fusion peptide of HRSV F when compared with other hydrophobic regions of the molecule.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.81715-0
2006-06-01
2019-10-13
Loading full text...

Full text loading...

/deliver/fulltext/jgv/87/6/1649.html?itemId=/content/journal/jgv/10.1099/vir.0.81715-0&mimeType=html&fmt=ahah

References

  1. Baker, K. A., Dutch, R. E., Lamb, R. A. & Jardetzky, T. S. ( 1999; ). Structural basis for paramyxovirus-mediated membrane fusion. Mol Cell 3, 309–319.[CrossRef]
    [Google Scholar]
  2. Bembridge, G. P., López, 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]
  3. Blasco, R. & Moss, B. ( 1995; ). Selection of recombinant vaccinia viruses on the basis of plaque formation. Gene 158, 157–162.[CrossRef]
    [Google Scholar]
  4. Bordo, D. & Argos, P. ( 1991; ). Suggestion for “safe” residue substitutions in site-directed mutagenesis. J Mol Biol 217, 721–729.[CrossRef]
    [Google Scholar]
  5. 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]
  6. 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 cultures and exhibits site-specific attenuation in the respiratory tract of mouse. J Virol 71, 8973–8982.
    [Google Scholar]
  7. 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]
  8. 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]
  9. Collins, P. L., Chanock, R. M. & Murphy, B. R. ( 2001; ). Respiratory syncytial virus. In Fields Virology, 4th edn, pp. 1443–1485. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
  10. Cross, K. J., Wharton, S. A., Skehel, J. J., Wiley, D. C. & Steinhauer, D. A. ( 2001; ). Studies on influenza haemagglutinin fusion peptide mutants generated by reverse genetics. EMBO J 20, 4432–4442.[CrossRef]
    [Google Scholar]
  11. 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]
  12. Falsey, A. R., Hennessey, P. A., Formica, M. A., Cox, C. & Walsh, E. E. ( 2005; ). Respiratory syncytial virus infection in elderly and high-risk adults. N Engl J Med 352, 1749–1759.[CrossRef]
    [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. Harter, C., James, P., Bachi, T., Semenza, G. & Brunner, J. ( 1989; ). Hydrophobic binding of the ectodomain of the influenza hemagglutinin to membranes occurs through the “fusion peptide”. J Biol Chem 264, 6459–6464.
    [Google Scholar]
  18. 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]
  19. Joshi, S. B., Dutch, R. E. & Lamb, R. A. ( 1998; ). A core trimer of the paramyxovirus fusion protein: parallels to influenza virus hemagglutinin and HIV-1 gp41. Virology 248, 20–34.[CrossRef]
    [Google Scholar]
  20. 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]
  21. Kyte, J. & Doolittle, R. ( 1982; ). A simple method for displaying the hydrophobic character of a protein. J Mol Biol 157, 105–132.[CrossRef]
    [Google Scholar]
  22. Lamb, R. A. ( 1993; ). Paramyxovirus fusion: a hypothesis for changes. Virology 197, 1–11.[CrossRef]
    [Google Scholar]
  23. Lamb, R. A. & Kolakofsky, D. ( 2001; ). Paramyxoviridae: the viruses and their replication. In Fields Virology, 4th edn, pp. 1305–1340. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
  24. 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]
  25. 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]
  26. 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]
  27. 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]
  28. 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]
  29. Ruiz-Argüello, M. B., Martín, D., Wharton, S. A. & 7 other authors ( 2004; ). Proteolytic cleavage of a soluble form of human respiratory syncytial virus fusion protein leads to exposure of the fusion peptide without changing the protein thermostability: implications for the membrane fusion mechanism. J Gen Virol 85, 3677–3687.[CrossRef]
    [Google Scholar]
  30. Russell, C. J., Jardetzky, T. & Lamb, R. A. ( 2004; ). Conserved glycine residues in the fusion peptide of the paramyxovirus fusion protein regulate activation of the native state. J Virol 78, 13727–13742.[CrossRef]
    [Google Scholar]
  31. Sergel, T. A., McGinnes, L. W. & Morrison, T. G. ( 2001; ). Mutations in the fusion peptide and adjacent heptad repeat inhibit folding or activity of the Newcastle disease virus fusion protein. J Virol 75, 7934–7943.[CrossRef]
    [Google Scholar]
  32. Skehel, J. J., Bayley, P. M., Brown, E. B., Martin, S. R., Waterfiels, M. D., White, J. M., Wilson, I. A. & Wiley, D. C. ( 1982; ). Changes in the conformation of influenza hemagglutinin at the pH optimum of virus-mediated membrane fusion. Proc Natl Acad Sci U S A 79, 968–972.[CrossRef]
    [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. Wrigley, N. G., Brown, E. B. & Skehel, J. J. ( 1986; ). Electron microscopy of influenza virus. In Electron Microscopy of Proteins, vol. 5, Viral Structure, pp. 103–163. Edited by J. R. Harris & R. W. Horne. London: Academic Press.
  37. 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]
  38. 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]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.81715-0
Loading
/content/journal/jgv/10.1099/vir.0.81715-0
Loading

Data & Media loading...

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error