1887

Abstract

The soluble form of the human respiratory syncytial virus (HRSV) attachment protein (Gs) was purified from the supernatant of infected cell cultures by immunoaffinity chromatography. Digestion of Gs with proteases and Western blot analysis identified two fragments that were partially resistant to protease degradation. Reactivity with diagnostic monoclonal antibodies located these two fragments in the primary structure of the G molecule. The large fragment spanned the C-terminal third of the G protein whereas the small fragment represented the N-terminal half of the large fragment. Purification of Gs from infected cells (either HEp-2 or M6) followed by protease digestion located host-cell-dependent glycosylation of the G protein in the unique part of the large protease-resistant fragment. The use of HRSV mutants encoding truncated G proteins allowed us to place some of the host-cell-dependent glycosylation differences in a small segment of the G protein. Interestingly, cell-specific glycosylations in the C-terminal half of the large protease-resistant fragment influenced the expression of certain epitopes located in its N-terminal half. These results bear important implications for the three-dimensional structure of the G glycoprotein.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-81-4-919
2000-04-01
2024-11-03
Loading full text...

Full text loading...

/deliver/fulltext/jgv/81/4/0810919a.html?itemId=/content/journal/jgv/10.1099/0022-1317-81-4-919&mimeType=html&fmt=ahah

References

  1. Anderson, L. J., Heirholzer, J. C., Tson, C., Hendry, R. M., Fernie, B. N., Stone, Y. & McIntosh, K. (1985). Antigenic characterization of respiratory syncytial virus strains with monoclonal antibodies. Journal of Infectious Diseases 151, 626-633.[CrossRef] [Google Scholar]
  2. Bembridge, G. P., Garcı́a-Beato, R., López, J. A., Melero, J. A. & Taylor, G. (1998). Subcellular site of expression and route of vaccination influence pulmonary eosinophilia following respiratory syncytial virus challenge in BALB/c mice sensitized to the attachment G protein. Journal of Immunology 161, 2473-2480. [Google Scholar]
  3. Cane, P. A. & Pringle, C. R. (1995). Evolution of subgroup A respiratory syncytial virus: evidence for progressive accumulation of amino acid changes in the attachment protein. Journal of Virology 69, 2918-2925. [Google Scholar]
  4. Cane, P. A., Matthews, D. A. & Pringle, C. R. (1991). Identification of variable domains of the attachment (G) protein of subgroup A respiratory syncytial viruses. Journal of General Virology 72, 2091-2096.[CrossRef] [Google Scholar]
  5. Collins, P. L., McIntosh, K. & Chanock, R. M. (1996). Respiratory syncytial virus. In Fields Virology, pp. 1313-1351. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott–Raven.
  6. Garcı́a, O., Martı́n, M., Dopazo, J., Arbiza, J., Frabasile, S., Russi, J., Hortal, M., Perez-Breña, P., Martı́nez, I., Garcı́a-Barreno, B. & Melero, J. A. (1994). Evolutionary pattern of human respiratory syncytial virus (subgroup A): cocirculating lineages and correlation of genetic and antigenic changes in the G glycoprotein. Journal of Virology 68, 5448-5459. [Google Scholar]
  7. Garcı́a-Barreno, B., Jorcano, J. L., Aukenbauer, T., López-Galı́ndez, C. & Melero, J. A. (1988). Participation of cytoskeletal intermediate filaments in the infectious cycle of human respiratory syncytial virus (RSV). Virus Research 9, 307-322.[CrossRef] [Google Scholar]
  8. Garcı́a-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. Journal of Virology 63, 925-932. [Google Scholar]
  9. Garcı́a-Barreno, B., Portela, A., Delgado, T., López, J. A. & Melero, J. A. (1990). Frame shift mutations as a novel mechanism for the generation of neutralisation resistant mutants of human respiratory syncytial virus. EMBO Journal 12, 4181-4187. [Google Scholar]
  10. Garcı́a-Barreno, B., Delgado, T., Akerlind-Stopner, B., Norrby, E. & Melero, J. A. (1992). Location of the epitope recognized by monoclonal antibody 63G on the primary structure of human respiratory syncytial virus G glycoprotein and the ability of synthetic peptides containing this epitope to induce neutralizing antibodies. Journal of General Virology 73, 2625-2630.[CrossRef] [Google Scholar]
  11. Garcı́a-Beato, R., Martı́nez, I., Francı́, C., Real, F. X., Garcı́a-Barreno, B. & Melero, J. A. (1996). Host cell effect upon glycosylation and antigenicity of human respiratory syncytial virus G glycoprotein. Virology 221, 301-309.[CrossRef] [Google Scholar]
  12. Hendrick, D. A., McIntosh, K. & Patterson, J. L. (1988). Further characterization of the soluble form of the G protein of respiratory syncytial virus. Journal of Virology 62, 2228-2233. [Google Scholar]
  13. Johnson, P. R., Spriggs, P. R., Olmsted, R. A. & Collins, P. L. (1987). The G glycoprotein of human respiratory syncytial viruses of subgroups A and B: extensive sequence divergence between antigenically related proteins. Proceedings of the National Academy of Sciences, USA 84, 5625-5629.[CrossRef] [Google Scholar]
  14. Lambert, D. M. (1988). Role of oligosaccharides in the structure and function of respiratory syncytial virus glycoproteins. Virology 130, 204-214. [Google Scholar]
  15. Langedijk, J. P. M., Schaaper, W. M. M., Meloen, R. H. & van Oirschot, J. T. (1996). Proposed three-dimensional model for the attachment protein G of respiratory syncytial virus. Journal of General Virology 77, 1249-1257.[CrossRef] [Google Scholar]
  16. Martı́nez, I., Dopazo, J. & Melero, J. A. (1997). Antigenic structure of the human respiratory syncytial virus G glycoprotein and relevance of hypermutation events for the generation of antigenic variants. Journal of General Virology 78, 2419-2429. [Google Scholar]
  17. Martı́nez, I., Valdés, O., Delfraro, A., Arbiza, J., Russi, J. & Melero, J. A. (1999). Evolutionary pattern of the G glycoprotein of human respiratory syncytial viruses from antigenic group B: the use of alternative termination codons and lineage diversification. Journal of General Virology 80, 125-130. [Google Scholar]
  18. Melero, J. A., Garcı́a-Barreno, B., Martı́nez, I., Pringle, C. R. & Cane, P. A. (1997). Antigenic structure, evolution and immunobiology of human respiratory syncytial virus attachment (G) protein. Journal of General Virology 78, 2411-2418. [Google Scholar]
  19. Moore, A. E., Sabachewsky, L. & Toolan, M. W. (1955). Culture characteristics of four permanent lines of human cancer cells. Cancer Research 15, 598-602. [Google Scholar]
  20. Mufson, M. A., Örvell, C., Rafnar, B. & Norrby, E. (1985). Two distinct subtypes of human respiratory syncytial virus. Journal of General Virology 66, 2111-2124.[CrossRef] [Google Scholar]
  21. Palomo, C., Garcı́a-Barreno, B., Peñas, C. & Melero, J. A. (1991). The G protein of human respiratory syncytial virus: significance of carbohydrate side-chains and the C-terminal end to its antigenicity. Journal of General Virology 72, 669-675.[CrossRef] [Google Scholar]
  22. Palomo, C., Cane, P. A. & Melero, J. A. (1999). Evaluation of the antibody specificities of human convalescent sera against the attachment (G) protein of human respiratory syncytial virus: influence of strain variation and carbohydrate side-chains. Journal of Medical Virology (in press).
  23. Roberts, S. R., Lichtenstein, D., Ball, L. A. & Wertz, G. W. (1994). The membrane-associated and secreted forms of the respiratory syncytial virus attachment glycoprotein G are synthesized from alternative initiation codons. Journal of Virology 68, 4538-4546. [Google Scholar]
  24. Rueda, P., Delgado, T., Portela, A., Melero, J. A. & Garcı́a-Barreno, B. (1991). Premature stop codons in the G glycoprotein of human respiratory syncytial viruses resistant to neutralization by monoclonal antibodies. Journal of Virology 65, 3374-3378. [Google Scholar]
  25. Rueda, P., Palomo, C., Garcı́a-Barreno, B. & Melero, J. A. (1995). The three C-terminal residues of human respiratory syncytial virus G glycoprotein (Long strain) are essential for integrity of multiple epitopes distinguishable by antiidiotypic antibodies. Viral Immunology 8, 37-46.[CrossRef] [Google Scholar]
  26. Sullender, W. M., Mufson, M. A., Anderson, L. J. & Wertz, G. W. (1991). Genetic diversity of the attachment protein of subgroup B respiratory syncytial virus. Journal of Virology 65, 5425-5434. [Google Scholar]
  27. Wertz, G. W., Collins, P. L., Huang, Y., Gruber, C., Levine, S. & Ball, L. A. (1985). Nucleotide sequence of the G protein of human respiratory syncytial virus reveals an unusual type of viral membrane. Proceedings of the National Academy of Sciences, USA 82, 4075-4079.[CrossRef] [Google Scholar]
/content/journal/jgv/10.1099/0022-1317-81-4-919
Loading
/content/journal/jgv/10.1099/0022-1317-81-4-919
Loading

Data & Media loading...

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