Advertisement

Analysis of Glycoproteins of Viruses in the Family Bunyaviridae

  • Xiaohong Shi
  • Richard M. Elliott
Part of the Methods in Molecular Biology book series (MIMB, volume 379)

Abstract

The membrane glycoproteins (Gn and Gc) of viruses in the family Bunyaviridae form projections on the virion envelope and are involved in virus entry and eliciting protective immunity. The glycoproteins are modified by N-linked glycosylation and accumulate in the Golgi complex where virions mature and bud. In this chapter, we describe the methods that have been used in our laboratory for the study of the glycoproteins of Bunyamwera virus, the prototype of the family. The protocols cover the expression of viral glycoproteins, examination of intracellular localization by immnunofluorescent confocal microscopy, radiolabeling, immunoprecipitation, and SDS-PAGE analysis of the proteins, and the improved reverse genetic system to rescue recombinant viruses that contain mutations at N-linked glycosylation sites.

Key Words

N-linked glycosylation Bunyaviridae Bunyamwera virus bunyavirus glycoprotein protein expression virus rescue and reverse genetics 

References

  1. 1.
    Elliott, R., Bouloy, M., Calisher, C. H., et al. (2000) Bunyaviridae, in Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses (van Regenmortel, M. H. V., Fauquet, C. M., Bishop, D. H. L., et al, eds.). Academic, San Diego, CA: pp. 599–621.Google Scholar
  2. 2.
    Calisher, C. H. (1996) History, Classification, and Taxonomy of Viruses in the Family Bunyaviridae. Plenum, New York & London.Google Scholar
  3. 3.
    Elliott, R. M. (1997) Emerging viruses: the Bunyaviridae. Mol. Med. 3, 572–577.PubMedGoogle Scholar
  4. 4.
    Pettersson, R. F. and Melin, L. (1997) Synthesis, assembly, and intracellular transport of Bunyaviridae membrane proteins, in The Bunyaviridae (Elliott, R. M., ed.). Plenum, New York, NY: pp. 159–188.Google Scholar
  5. 5.
    Shi, X. and Elliott, R. M. (2004) Analysis of N-linked glycosylation of hantaan virus glycoproteins and the role of oligosaccharide side chains in protein folding and intracellular trafficking. J. Virol. 78, 5414–5422.CrossRefPubMedGoogle Scholar
  6. 6.
    Shi, X., Lappin, D. F., and Elliott, R. M. (2004) Mapping the Golgi targeting and retention signal of Bunyamwera virus glycoproteins. J. Virol. 78, 10,793–10,802.CrossRefPubMedGoogle Scholar
  7. 7.
    Madoff, D. H. and Lenard, J. (1982). A membrane glycoprotein that accumulates intracellularly: cellular processing of the large glycoprotein of LaCrosse virus. Cell 28, 821–829.CrossRefPubMedGoogle Scholar
  8. 8.
    Pesonen, M., Ronnholm, R., Kuismanen, E., and Pettersson, R. F. (1982) Characterization of the oligosaccharides of Inkoo virus envelope glycoproteins. J. Gen. Virol. 63, 425–434.CrossRefPubMedGoogle Scholar
  9. 9.
    Cash, P., Hendershot, L., and Bishop, D. H. (1980) The effect of glycosylation inhibitors on the maturation and intracellular polypeptide synthesis induced by snowshoe hare bunyavirus. Virology 103, 235–240.CrossRefPubMedGoogle Scholar
  10. 10.
    Schmaljohn, C. S., Hasty, S. E., Rasmussen, L., and Dalrymple, J. M. (1986) Hantaan virus replication: effects of monensin, tunicamycin and endoglycosidases on the structural glycoproteins. J. Gen. Virol. 67, 707–717.CrossRefPubMedGoogle Scholar
  11. 11.
    Ruusala, A., Persson, R., Schmaljohn, C. S., and Pettersson, R. F. (1992) Coexpression of the membrane glycoproteins G1 and G2 of Hantaan virus is required for targeting to the Golgi complex. Virology 186, 53–64.CrossRefPubMedGoogle Scholar
  12. 12.
    Nakitare, G. W. and Elliott, R. M. (1993) Expression of the Bunyamwera virus M genome segment and intracellular localization of NSm. Virology 195, 511–520.CrossRefPubMedGoogle Scholar
  13. 13.
    Salanueva, I. J., Novoa, R. R., Cabezas, P., et al. (2003) Polymorphism and structural maturation of bunyamwera virus in Golgi and post-Golgi compartments. J. Virol. 77, 1368–1381.CrossRefPubMedGoogle Scholar
  14. 14.
    Shi, X. and Elliott, R. M. (2002) Golgi localization of Hantaan virus glycoproteins requires coexpression of Gl and G2. Virology 300, 31–38.CrossRefPubMedGoogle Scholar
  15. 15.
    Maddon, P. J., Dalgleish, A. G., McDougal, J. S., Clapham, P. R., Weiss, R. A., and Axel, R. (1986) The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell 47, 333–348.CrossRefPubMedGoogle Scholar
  16. 16.
    Buchholz, U. J., Finke, S., and 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.PubMedGoogle Scholar
  17. 17.
    Fuerst, T. R., Niles, E. G., Studier, F. W., and Moss, B. (1986) Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 83, 8122–8126.CrossRefPubMedGoogle Scholar
  18. 18.
    Watret, G. E., Pringle, C. R., and Elliott, R. M. (1985) Synthesis of bunyavirus-specific proteins in a continuous cell line (XTC-2) derived from Xenopus laevis. J. Gen. Virol. 66(Pt 3), 473–482.CrossRefPubMedGoogle Scholar
  19. 19.
    Lappin, D. F., Nakitare, G. W., Palfreyman, J. W., and Elliott, R. M. (1994) Localization of Bunyamwera bunyavirus G1 glycoprotein to the Golgi requires association with G2 but not with NSm. J. Gen. Virol. 75(Pt 12), 3441–3451.CrossRefPubMedGoogle Scholar
  20. 20.
    Nakamura, N., Rabouille, C., Watson, R., et al. (1995) Characterization of a cis-Golgi matrix protein, GM130. J. Cell Biol. 131, 1715–1726.CrossRefPubMedGoogle Scholar
  21. 21.
    Griffith, K. J., Chan, E. K., Lung, C. C., et al. (1997) Molecular cloning of a novel 97-kd Golgi complex autoantigen associated with Sjogren’s syndrome. Arthritis Rheum. 40, 1693–1702.CrossRefPubMedGoogle Scholar
  22. 22.
    Bridgen, A. and Elliott, R. M. (1996) Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs. Proc. Natl. Acad. Sci. USA 93, 15,400–15,404.CrossRefPubMedGoogle Scholar
  23. 23.
    Shi, X., Brauburger, K., and Elliott, R. M. (2005) Role of N-linked glycans on Bunyamwera virus glycoproteins in intracellular trafficking, protein folding, and virus infectivity. J. Virol. 79, 13,725–13,734.CrossRefPubMedGoogle Scholar
  24. 24.
    Moss, B., Elroy-Stein, O., Mizukami, T., Alexander, W. A., and Fuerst, T. R. (1990) Product review. New mammalian expression vectors. Nature 348, 91–92.CrossRefPubMedGoogle Scholar
  25. 25.
    Briese, T., Rambaut, A., and Lipkin, W. I. (2004) Analysis of the medium (M) segment sequence of Guaroa virus and its comparison to other orthobunyaviruses. J. Gen. Virol. 85, 3071–3077.CrossRefPubMedGoogle Scholar
  26. 26.
    Lowen, A. C., Noonan, C., McLees, A., and Elliott, R. M. (2004) Efficient bunyavirus rescue from cloned cDNA. Virology 330, 493–500.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Xiaohong Shi
    • 1
  • Richard M. Elliott
    • 1
  1. 1.Centre for Biomolecular Sciences, School of BiologyUniversity of St AndrewsSt AndrewsUK

Personalised recommendations