Interaction Between Respiratory Syncytial Virus and Glycosaminoglycans, Including Heparan Sulfate

  • Louay K. Hallak
  • Steven A. Kwilas
  • Mark E. Peeples
Part of the Methods in Molecular Biology book series (MIMB, volume 379)


Glycosaminoglycans (GAGs), including heparan sulfate (HS), are expressed on the surface of nearly all cells, linked to transmembrane proteins. These GAGs are sulfated to varying extents, lending a negative charge, and are used by a large number of viruses to initiate infection of immortalized cell lines. Here we describe the rationale and methods for analyzing GAG usage by one such virus, respiratory syncytial virus (RS V). The protocols presented allow the determination of which GAG(s) is employed by the virus, which GAG modification(s) is important, and whether the important GAG is on the cell or on the virus. We also discuss the finding that many viruses are selected for GAG usage during passage in culture and present a method for rapidly determining whether GAG usage is characteristic of a wild virus or is limited to laboratory-adapted virus.

Key Words

Glycosaminoglycans heparan sulfate heparin chondroitin sulfate proteoglycan respiratory syncytial virus RSV GAG paramyxovirus sulfation 


  1. 1.
    Yamada, T. and Kawasaki, T. (2005) Microbial synthesis of hyaluronan and chitin: new approaches. J. Biosci. Bioeng. 99, 521–528.CrossRefPubMedGoogle Scholar
  2. 2.
    Ajit, V., Richard, C., Jeffrey, E., et al. (2002) The Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  3. 3.
    Kusche-Gullberg, M. and Kjellen, L. (2003) Sulfotransferases in glycosaminoglycan biosynthesis. Curr. Opin. Struct. Biol. 13, 605–611.CrossRefPubMedGoogle Scholar
  4. 4.
    Burstein, M. (1952) [The antithrombin effect of heparin; role of heparin concentration.]. C.R. Seances Soc. Biol. Fil. 146, 641–642.PubMedGoogle Scholar
  5. 5.
    Sasisekharan, R., Ernst, S., and Venkataraman, G. (1997) On the regulation of fibroblast growth factor activity by heparin-like glycosaminoglycans. Angiogenesis. 1, 45–54.CrossRefPubMedGoogle Scholar
  6. 6.
    Taylor, K. R., Rudisill, J. A., and Gallo, R. L. (2005) Structural and sequence motifs in dermatan sulfate for promoting fibroblast growth factor-2 (FGF-2) and FGF-7 activity. J. Biol. Chem. 280, 5300–5306.CrossRefPubMedGoogle Scholar
  7. 7.
    Laterra, J., Silbert, J. E., and Culp, L. A. (1983) Cell surface heparan sulfate mediates some adhesive responses to glycosaminoglycan-binding matrices, including fibronectin. J. Cell Biol. 96, 112–123.CrossRefPubMedGoogle Scholar
  8. 8.
    Baldassarri, L., Bertuccini, L., Creti, R., et al. (2005) Glycosaminoglycans mediate invasion and survival of Enterococcus faecalis into macrophages. J. Infect. Dis. 191, 1253–1262.CrossRefPubMedGoogle Scholar
  9. 9.
    Frick, I. M., Schmidtchen, A., and Sjobring, U. (2003). Interactions between M proteins of Streptococcus pyogenes and glycosaminoglycans promote bacterial adhesion to host cells. Fur. J. Biochem. 270, 2303–2311.Google Scholar
  10. 10.
    Henry-Stanley, M. J., Hess, D. J., Erlandsen, S. L., and Wells, C. L. (2005). Ability of the heparan sulfate proteoglycan syndecan-1 to participate in bacterial translocation across the intestinal epithelial barrier. Shock 24, 571–576.CrossRefPubMedGoogle Scholar
  11. 11.
    Menozzi, F. D., Pethe, K., Bifani, P., Soncin, F., Brennan, M. J., and Locht, C. (2002) Enhanced bacterial virulence through exploitation of host glycosaminoglycans. Mol. Microbiol. 43, 1379–1386.CrossRefPubMedGoogle Scholar
  12. 12.
    van Putten, J. P., Duensing, T. D., and Cole, R. L. (1998) Entry of OpaA+ gonococci into HEp-2 cells requires concerted action of glycosaminoglycans, fibronectin and integrin receptors. Mol. Microbiol. 29, 369–379.CrossRefPubMedGoogle Scholar
  13. 13.
    Varez-Dominguez, C., Vazquez-Boland, J. A., Carrasco-Marin, E., Lopez-Mato, P., and Leyva-Cobian, F. (1997) Host cell heparan sulfate proteoglycans mediate attachment and entry of Listeria monocytogenes, and the listerial surface protein ActA is involved in heparan sulfate receptor recognition. Infect. Immun. 65, 78–88.Google Scholar
  14. 14.
    Barth, H., Schafer, C., Adah, M. L, et al. (2003) Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate. J. Biol. Chem. 278, 41,003–41,012.CrossRefPubMedGoogle Scholar
  15. 15.
    Bousarghin, L., Hubert, P., Franzen, E., Jacobs, N., Boniver, J., and Delvenne, P. (2005) Human papillomavirus 16 virus-like particles use heparan sulfates to bind dendritic cells and colocalize with langerin in Langerhans cells. J. Gen. Virol. 86, 1297–1305.CrossRefPubMedGoogle Scholar
  16. 16.
    Byrnes, A. P. and Griffin, D. E. (1998). Binding of Sindbis virus to cell surface heparan sulfate. J. Virol. 72, 7349–7356.PubMedGoogle Scholar
  17. 17.
    Escribano-Romero, E., Jimenez-Clavero, M. A., Gomes, P., Garcia-Ranea, J. A., and Ley, V. (2004) Heparan sulphate mediates swine vesicular disease virus attachment to the host cell. J. Gen. Virol. 85, 653–663.CrossRefPubMedGoogle Scholar
  18. 18.
    Feldman, S. A., Audet, S., and Beeler, J. A. (2000). The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate. J. Virol. 74, 6442–6447.CrossRefPubMedGoogle Scholar
  19. 19.
    Germi, R., Crance, J. M., Garin, D., et al. (2002) Heparan sulfate-mediated binding of infectious dengue virus type 2 and yellow fever virus. Virology 292, 162–168.CrossRefPubMedGoogle Scholar
  20. 20.
    Hilgard, P. and Stocken, R. (2000) Heparan sulfate proteoglycans initiate dengue virus infection of hepatocytes. Hepatology 32, 1069–1077.CrossRefPubMedGoogle Scholar
  21. 21.
    Hulst, M. M., van Gennip, H. G., Vlot, A. C., Schooten, E., de Smit, A. J., and Moormann, R. J. (2001) Interaction of classical swine fever virus with membrane-associated heparan sulfate: role for virus replication in vivo and virulence. J. Virol. 75, 9585–9595.CrossRefPubMedGoogle Scholar
  22. 22.
    Jackson, T., Ellard, F. M., Ghazaleh, R. A., et al. (1996) Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate. J. Virol. 70, 5282–5287.PubMedGoogle Scholar
  23. 23.
    Jones, K. S., Petrow-Sadowski, C., Bertolette, D. C., Huang, Y., and Ruscetti, F. W. (2005). Heparan sulfate proteoglycans mediate attachment and entry of human T-cell leukemia virus type 1 virions into CD4+ T cells. J. Virol. 79, 12,692–12,702.CrossRefPubMedGoogle Scholar
  24. 24.
    Kroschewski, H., Allison, S. L., Heinz, F. X., and Mandl, C. W. (2003) Role of heparan sulfate for attachment and entry of tick-borne encephalitis virus. Virology 308, 92–100.CrossRefPubMedGoogle Scholar
  25. 25.
    Rue, C. A. and Ryan, P. (2002) Characterization of pseudorabies virus glycoprotein C attachment to heparan sulfate proteoglycans. J. Gen. Virol. 83, 301–309.PubMedGoogle Scholar
  26. 26.
    WuDunn, D. and Spear, P. G. (1989). Initial interaction of herpes simplex virus with cells is binding to heparan sulfate. J. Virol. 63, 52–58.PubMedGoogle Scholar
  27. 27.
    O’ Donnell, C. D., Tiwari, V., Oh, M. J., and Shukla, D. (2005) A role for heparan sulfate 3-O-sulfotransferase isoform 2 in herpes simplex virus type 1 entry and spread. Virology 346, 452–459.CrossRefPubMedGoogle Scholar
  28. 28.
    Klimstra, W. B., Ryman, K. D., and Johnston, R. E. (1998) Adaptation of Sindbis virus to BHK cells selects for use of heparan sulfate as an attachment receptor. J. Virol. 72, 7357–7366.PubMedGoogle Scholar
  29. 29.
    Mandl, C. W., Kroschewski, H., Allison, S. L., et al. (2001) Adaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo. J. Virol. 75, 5627–5637.CrossRefPubMedGoogle Scholar
  30. 30.
    Bourgeois, C., Bour, J. B., Lidholt, K., Gauthray, C., and Pothier, P. (1998) Heparin-like structures on respiratory syncytial virus are involved in its infectivity in vitro. J. Virol. 72, 7221–7227.PubMedGoogle Scholar
  31. 31.
    Feldman, S. A., Crim, R. L., Audet, S. A., and Beeler, J. A. (2001) Human respiratory syncytial virus surface glycoproteins F, G and SH form an oligomeric complex. Arch. Virol. 146, 2369–2383.CrossRefPubMedGoogle Scholar
  32. 32.
    Hallak, L., Spillman, D., Collins, P. L., and Peeples, M. E. (2000) Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection. J. Virol. 74, 10,508–10,513.CrossRefPubMedGoogle Scholar
  33. 33.
    Hallak, L. K., Collins, P. L., Knudson, W., and Peeples, M. E. (2000) Iduronic acid-containing glycosaminoglycans on target cells are required for efficient respiratory syncytial virus infection. Virology 271, 264–275.CrossRefPubMedGoogle Scholar
  34. 34.
    Krusat, T. and Streckert, H. J. (1997). Heparin-dependent attachment of respiratory syncytial virus (RSV) to host cells. Arch. Virol. 142, 1247–1254.CrossRefPubMedGoogle Scholar
  35. 35.
    Shields, B., Mills, J., Ghildyal, R., Gooley, P., and Meanger, J. (2003) Multiple heparin binding domains of respiratory syncytial virus G mediate binding to mammalian cells. Arch.Virol. 148, 1987–2003.CrossRefPubMedGoogle Scholar
  36. 36.
    Techaarpornkul, S., Collins, P. L., and Peeples, M. E. (2002). Respiratory syncytial virus with the fusion protein as its only viral glycoprotein is less dependent on cellular glycosaminoglycans for attachment than complete virus. Virology 294, 296–304.CrossRefPubMedGoogle Scholar
  37. 37.
    Nakazawa, K., Morita, A., Nakano, H., Mano, C., and Tozawa, N. (1996). Keratan sulfate synthesis by corneal stromal cells within three-dimensional collagen gel cultures. J. Biochem. (Tokyo) 120, 117–125.Google Scholar
  38. 38.
    Resch, M. D., Nagy, Z. Z., Szentmary, N., Mathe, M., Kovalszky, I., and Suveges, I. (2005) Spatial distribution of keratan sulfate in the rabbit cornea following photorefractive keratectomy. J. Refract. Surg. 21, 485–493.PubMedGoogle Scholar
  39. 39.
    Zhang, Y., Conrad, A. H., Tasheva, E. S., et al. (2005) Detection and quantification of sulfated disaccharides from keratan sulfate and chondroitin/dermatan sulfate during chick corneal development by ESI-MS/MS. Invest. Ophthalmol. Vis. Sci. 46, 1604–1614.CrossRefPubMedGoogle Scholar
  40. 40.
    Wendel, M., Sommarin, Y., and Heinegard, D. (1998) Bone matrix proteins: isolation and characterization of a novel cell-binding keratan sulfate proteoglycan (osteoadherin) from bovine bone. J. Cell Biol. 141, 839–847.CrossRefPubMedGoogle Scholar
  41. 41.
    Knox, S., Fosang, A. J., Last, K., Melrose, J., and Whitelock, J. (2005) Perlecan from human epithelial cells is a hybrid heparan/chondroitin/keratan sulfate proteoglycan. FEES Lett. 579, 5019–5023.CrossRefGoogle Scholar
  42. 42.
    Zhang, L., Peeples, M. E., Boucher, R. C., Collins, P. L., and Pickles, R. J. (2002) Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology. J. Virol. 76, 5654–5666.CrossRefPubMedGoogle Scholar
  43. 43.
    Coster, L. and Fransson, L. A. (1981) Isolation and characterization of dermatan sulphate proteoglycans from bovine sciera. Biochem. J. 193, 143–153.PubMedGoogle Scholar
  44. 44.
    Malstrom, A., Carlstedt, I., Aberg, L., and Fransson, L. A. (1975) The copolymeric structure of dermatan sulphate produced by cultured human fibroblasts. Different distribution of iduronic acid and glucuronic acid-containing units in soluble and cell-associated glycans. Biochem. J. 151, 477–489.PubMedGoogle Scholar
  45. 45.
    Esko, J. D., Elgavish, A., Prasthofer, T., Taylor, W. H., and Weinke, J. L. (1986) Sulfate transport-deficient mutants of Chinese hamster ovary cells. Sulfation of glycosaminoglycans dependent on cysteine. J. Biol. Chem. 261, 15,725–15,733.PubMedGoogle Scholar
  46. 46.
    Esko, J. D., Stewart, T. E., and Taylor, W. H. (1985). Animal cell mutants defective in glycosaminoglycan biosynthesis. Proc. Natl. Acad. Sci. USA 82, 3197–3201.CrossRefPubMedGoogle Scholar
  47. 47.
    Esko, J. D., Weinke, J. L., Taylor, W. H., et al. (1987) Inhibition of chondroitin and heparan sulfate biosynthesis in Chinese hamster ovary cell mutants defective in galactosyltransferase I. J. Biol. Chem. 262, 12,189–12,195.PubMedGoogle Scholar
  48. 48.
    Chen, Y., Maguire, T., Hileman, R. E., et al. (1997). Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate [see comments], Nat. Med. 3, 866–871.CrossRefPubMedGoogle Scholar
  49. 49.
    Mondor, I., Ugolini, S., and Sattentau, Q. J. (1998). Human immunodeficiency virus type 1 attachment to HeLa CD4 cells is CD4 independent and gpl20 dependent and requires cell surface heparans. J. Virol. 72, 3623–3634.PubMedGoogle Scholar
  50. 50.
    Zhao, Q., Pacheco, J. M., and Mason, P. W. (2003) Evaluation of genetically engineered derivatives of a Chinese strain of foot-and-mouth disease virus reveals a novel cell-binding site which functions in cell culture and in animals. J. Virol. 77, 3269–3280.CrossRefPubMedGoogle Scholar
  51. 51.
    Heil, M. L., Albee, A., Strauss, J. H., and Kuhn, R. J. (2001) An amino acid substitution in the coding region of the E2 glycoprotein adapts Ross River virus to utilize heparan sulfate as an attachment moiety. J. Virol. 75, 6303–6309.CrossRefPubMedGoogle Scholar
  52. 52.
    Techaarpornkul, S., Barretto, N., and 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.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Louay K. Hallak
    • 1
  • Steven A. Kwilas
    • 1
    • 2
  • Mark E. Peeples
    • 1
    • 3
  1. 1.Center for Vaccines and ImmunityColumbus Children’s Research InstituteColumbus
  2. 2.Department of Immunology/MicrobiologyRush UniversityChicago
  3. 3.Department of PediatricsThe Ohio State University College of MedicineColumbus

Personalised recommendations