Advertisement

The Use of Monoclonal Antibodies and Lectins to Identify Changes in Viral Glycoproteins That are Influenced by Glycosylation

The Case of Human Respiratory Syncytial Virus Attachment (G) Glycoprotein
  • Joanna Rawling
  • José A. Melero
Part of the Methods in Molecular Biology book series (MIMB, volume 379)

Abstract

The influence of viral envelope glycans is often overlooked, but one should bear in mind that variable glycosylation may affect the properties of viral envelope glycoproteins and potentially alter the course of an infection. Hence, there is a need for simple methods that can be use to identify changes in the glycosylation pattern of viral glycoproteins in a large number of samples. We describe here methods for the analysis of cellline specific changes in glycosylation of the respiratory syncytial virus (RSV) attachment glycoprotein (G), which involve the use of lectins and anti-carbohydrate antibodies. Given the role of the G glycoprotein in RSV antigenicity, we also describe procedures based on Western blotting to determine the effect of G protein glycosylation changes on reactivity with human sera. We found that glycosylation of the C-terminal domain of the G protein reduces reactivity with human sera, indicating that variable glycosylation may contribute to evasion of the humoral immune response by RSV.

Key Words

Respiratory syncytial virus G protein lectin V8 protease 

References

  1. 1.
    Kornfeld, R. and Kornfeld, S. (1985) Assembly of asparagine-linked oligosaccharides. Ann. Rev. Biochem. 54, 631–664.CrossRefPubMedGoogle Scholar
  2. 2.
    Hansen, J. E., Lund, O., Tolstrup, N., Gooley, A. A., Williams, K. L., and Brunak, S. (1998) NetOglyc: Prediction of mucin type O-glycosylation sites based on sequence content and surface accesibility. Glycoconj. J. 15, 115–130.CrossRefPubMedGoogle Scholar
  3. 3.
    Collins, P. L., Cannock, R. M., and Murphy, B. R. (2001) Respiratory syncytial virus, in Field’s Virology, 4th Ed. Lippincott, Wiliams and Wilkins, Philadelphia, PA: pp. 1443–1485.Google Scholar
  4. 4.
    Korber, B., Gaschen, B., Yusim, K., Thakallapally, R., Kesmir, C., and Detours, V. (2001) Evolutionary and immunological implications of contemporary HIV-1 variation. Br. Med. Bull. 58, 19–42.CrossRefPubMedGoogle Scholar
  5. 5.
    Jentoft, N. (1990) Why are proteins O-glycosylated? Trends Biochem. Sci. 15, 291–294.CrossRefPubMedGoogle Scholar
  6. 6.
    Sanders, R. W., Venturi, M., Schiffner, L., et al. (2002) The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120. J. Virol. 76, 7923–8305.CrossRefGoogle Scholar
  7. 7.
    Herbert, D. N., Zhang, J. X., Chen, W., Foellmer, B., and Helenius, A. (1997) The number and location of glycans on influenza hemagglutinin determine folding and association with calnexin and calreticulin. J. Cell Bio. 139, 613–623.CrossRefGoogle Scholar
  8. 8.
    Wagner, R., Heuer, D., Wolff, T., Herwig, A., and Klenk, H-D. (2002) N-glycans attached to the stem domain of haemagglutinin efficiently regulate influenza A virus replication. J. Gen. Virol. 83, 601–609.PubMedGoogle Scholar
  9. 9.
    Hart, M. L., Saifuddin, M., and Spear G. T. (2003) Glycosylation inhibitors and neuraminidase enhance human immunodeficiency virus type 1 binding and neutralization by mannose-binding lectin. J. Gen. Virol. 84, 358–360.CrossRefGoogle Scholar
  10. 10.
    Lin, G., Simmons, G., Pohlmann, S., et al. (2003) Differential N-linked glycosylation of human immunodeficiency virus and Ebola virus envelope glycoproteins modulates interactions with DC-SIGN and DC-SIGNR. J. Virol. 11, 1337–1346.CrossRefGoogle Scholar
  11. 11.
    Kaverin, N. V., Rudneva, I. A., Ilyushina, N. A., et al. (2002) Structure of antigenic sites on the hemagglutinin molecule of H5 avian influenza virus and phenotypic variation of escape mutants. J. Gen. Virol. 83, 2497–2505.PubMedGoogle Scholar
  12. 12.
    Melero, J. A., García-Barreno, B., Martínez, I., Pringle, C. R., and Cane, P. A. (1997) Antigenic structure, evolution and immunobiology of human respiratory syncytial virus attachment (G) protein. J. Gen. Virol. 78, 2411–2418.PubMedGoogle Scholar
  13. 13.
    Ohuchi, M., Ohuchi, R., Feldmann, A., and Klenk, H-D. (1997) Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety. J. Virol. 71, 8377–8384.PubMedGoogle Scholar
  14. 14.
    Kawaoka, Y., Naeve, C. W., and Webster, R. G. (1984) Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? Virology 139, 303–316.CrossRefPubMedGoogle Scholar
  15. 15.
    Cole, K. S., Steckbeck, J. D., Rowles, J. L., Desroisers, R. C., and Montelaro, R. C. (2004) Removal of N-linked glycosylation sites in the VI region of simian immunodeficiency virus gp120 results in redirection of B-cell responses to V3. J. Virol. 78, 1525–1539.CrossRefPubMedGoogle Scholar
  16. 16.
    Botarelli, P., Houlden, B. A., Haigwood, N. L., Servis, C., Montagna, D., and Abrignani, S. (1991) N-glycosylation of HIV gp120 may constrain recognition by T lymphocytes. J. Immunol. 147, 3128–3132PubMedGoogle Scholar
  17. 17.
    Alexander, S., and Elder, J. H. (1984) Carbohydrate dramatically influences immune reactivity of antisera to viral glycoprotein antigens. Science 226, 1328–1330.CrossRefPubMedGoogle Scholar
  18. 18.
    Wiley, R. L., Shibata, R., Freed, E. O., Cho, M. W., and Martin, M. A. (1996) Differential glycosylation, virion incorporation, and sensitivity to neutralizing antibodies of human immunodeficiency virus type 1 envelope produced from infected primary T-lymphocyte and macrophage cultures. J. Virol. 70, 6431–6436.Google Scholar
  19. 19.
    Harvey, D. J. (2001) Identification of protein-bound carbohydrates by mass spectrometry. Proteomics 1, 311–328.CrossRefPubMedGoogle Scholar
  20. 20.
    Taylor-Papadimitrou, J., Burchell, J., Miles, D. W., and Dalziel, M. (1999) MUC1 and cancer. Biochim. Biophys. Acta 1455, 301–313.Google Scholar
  21. 21.
    Wertz, G. W., Collins, P. L., Hang, Y., Gruber, C., Levine, S., and Ball, L. A. (1985) Nucleotide sequence of the G protein of human respiratory syncytial virus reveals an unusual type of membrane protein. Proc. Natl. Acad. Sci. USA 82, 4075–4079.CrossRefPubMedGoogle Scholar
  22. 22.
    Wilson, I. A., Skehel, J. J., and Wiley, D. C. (1981) Structure of the hemagglutinin membrane glyccoprotein of influenza virus at 3 Å resolution. Nature 289, 366–373.CrossRefPubMedGoogle Scholar
  23. 23.
    Ogert, R. A., Lee, M. K., Ross, W., Buckler-White, A., Martin, M. A., and Cho, M. W. (2001) N-linked glycosylation sites adjacent to and within the V2 and the V3 loops of dualtropic human immunodeficiency virus type 1 isolate DH12 gp120 affect coreceptor usage and cellular tropism. J. Virol. 75, 5998–6006.CrossRefPubMedGoogle Scholar
  24. 24.
    Wei, X., Decker, J. M., Wang, S., et al. (2003) Antibody neutralization and escape by HIV-1. Nature 422, 307–312.CrossRefPubMedGoogle Scholar
  25. 25.
    Skehel, J. J. and Wiley, D. C. (2000) Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Ann. Rev. Biochem. 69, 531–569.CrossRefPubMedGoogle Scholar
  26. 26.
    Collins, P. L. and Mottet, G (1992) Oligomerization and post-translational processing of glycoprotein G of human respiratory syncytial virus: altered O-glycosylation in the presence of brefeldin A. J. Gen. Virol. 73, 849–863.CrossRefPubMedGoogle Scholar
  27. 27.
    Roberts, S. R., Lichtenstein, D., Ball, L. A., and Wertz, G. W. (1994) The membrane-associated and secreted forms of the respiratory syncytial virus attachment glycoprotein are synthesized from alternative initiation codons. J. Virol. 68, 4538–4546.PubMedGoogle Scholar
  28. 28.
    Escribano-Romero, E., Rawling, J., García-Barreno, B., and Melero J. A. (2004) The soluble form of human respiratory syncytial virus attachment protein differs from the membrane-bound form in its oligomeric state but is still capable of binding to cell surface proteoglycans. J. Virol. 78, 3524–3532.CrossRefPubMedGoogle Scholar
  29. 29.
    Martínez, I., Dopazo, J., and 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. J. Gen. Virol. 78, 2419–2429PubMedGoogle Scholar
  30. 30.
    Palomo, C., Cane, P. A., and Melero, J. A. (2000) Evaluation of the antibody specificities of human convalescent-phase sera against the attachment (G) protein of human respiratory syncytial virus: influence of strain variation and carbohydrate side chains. J. Med. Virol. 60, 468–474.CrossRefPubMedGoogle Scholar
  31. 31.
    Zlateva, K. T., Lemey, P., Vandamme, A. M., and Van Ranst, M. (2004) Molecular evolution and circulation patterns of human respiratory syncytial virus subgroup a: positively selected sites in the attachment G glycoprotein. J. Virol. 78, 4675–4683.CrossRefPubMedGoogle Scholar
  32. 32.
    García-Beato, R., Martinez, I., Franci, C., Real, F. X., Garcia-Barreno, B., and Melero, J. A. (1996) Host cell effect upon glycosylation and antigenicity of human respiratory syncytial virus G glycoprotein. Virology 221, 301–309.CrossRefPubMedGoogle Scholar
  33. 33.
    Cane, P., Thomas, H. M., Simpson, A. F., Evans, J. E., Hart, C. A., and Pringle, C. R. (1996) Analysis of the human serological response to a variable region of the attachment (G) protein of respiratory syncytial virus during primary infection. J. Med. Virol. 48, 253–261.CrossRefPubMedGoogle Scholar
  34. 34.
    García, O., Martín, M., Dopazo, J., et al. (1994) Evolutionary pattern of human respiratory syncytial virus (subgroup A): cocirculating lineages and correlation of genetic and antigenic changes in the G glycoprotein. J. Virol. 68, 5448–5459.PubMedGoogle Scholar
  35. 35.
    Hanski, C., Bornhoeft, G., Topf, N., Hermann, U., Stein, H., and Riecken, E-O. (1990) Detection of a mucin marker for the adenoma-carcinoma sequence in the human colonic mucosa by monoclonal antibody AM-3. J. Clin. Pathol. 43, 379–385.CrossRefPubMedGoogle Scholar
  36. 36.
    Fukushi, Y., Hakomori, S., and Shelard, T. (1984) Location and alteration of mono-, di-, and trifucosyl a1-3type 2chain structures during human embryogenesis and human cancer. J. Exp. Med. 159, 506–520.CrossRefGoogle Scholar
  37. 37.
    de Bolós, C., Garrido, M., and Real, F. X. (1995) MUC6 apomucin shows a distinct normal tissue distribution that correlates with Lewis antigen expression in the human stomach. Gastroenterology 109, 723–734.CrossRefPubMedGoogle Scholar
  38. 38.
    Sakamoto, J., Furukawa, K., Cordon-Cardo, C., et al. (1986) Expression of Lewis a, Lewis b and Y blood group antigens in human tumour-derived cell lines. Cancer Res. 46, 1553–1561.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Joanna Rawling
    • 1
  • José A. Melero
    • 1
  1. 1.Biología ViralCentro Nacional de Microbiología, Instituto de Salud Carlos III, MajadahondaMadridSpain

Personalised recommendations