Advertisement

Expression and Purification of Viral Glycoproteins Using Recombinant Vaccinia Viruses for Functional and Structural Studies

  • Zhu-Nan Li
  • David A. Steinhauer
Part of the Methods in Molecular Biology book series (MIMB, volume 379)

Abstract

Methods for generating recombinant vaccinia viruses for the expression of foreign viral glycoproteins in mammalian cell lines and the purification of expressed viral glycoproteins are described. These methods are based on many years of experience with the influenza hemagglutinin glycoprotein (HA). However, they are applicable for studies on other viral glycoproteins, and with slight modifications, could be useful for cellular proteins as well.

Key Words

Viral glycoproteins recombinant vaccinia viruses influenza hemagglutinin structure function 

References

  1. 1.
    Mackett, M., Smith, G. L., and Moss, B. (1982) Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc. Natl. Acad. Sci. USA 79, 7415–7419.CrossRefPubMedGoogle Scholar
  2. 2.
    Moss, B. (1996) Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc. Natl. Acad. Sci. USA 93, 11,341–11,348.CrossRefPubMedGoogle Scholar
  3. 3.
    Blasco, R. and Moss, B. (1995) Selection of recombinant vaccinia viruses on the basis of plaque formation. Gene 158, 157–162.CrossRefPubMedGoogle Scholar
  4. 4.
    Blasco, R. and Moss, B. (1991) Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer envelope protein. J. Virol. 65, 5910–5920.PubMedGoogle Scholar
  5. 5.
    Cross, K. J., Wharton, S. A., Skehel, J. J., Wiley, D. C., and Steinhauer, D. A. (2001) Studies on influenza haemagglutinin fusion peptide mutants generated by reverse genetics. EMBO J. 20, 4432–4442.CrossRefPubMedGoogle Scholar
  6. 6.
    Martin, J., Wharton, S. A., Lin, Y. P., et al. (1998) Studies of the binding properties of influenza hemagglutinin receptor-site mutants. Virology 241, 101–111.CrossRefPubMedGoogle Scholar
  7. 7.
    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
  8. 8.
    Chen, J., Lee, K. H., Steinhauer, D. A., Stevens, D. J., Skehel, J. J., and Wiley, D. C. (1998) Structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation. Cell 95, 409–417.CrossRefPubMedGoogle Scholar
  9. 9.
    Gamblin, S. J., Haire, L. F., Russell, R. J., et al. (2004) The structure and receptor binding properties of the 1918 influenza hemagglutinin. Science 303, 1838–1842.CrossRefPubMedGoogle Scholar
  10. 10.
    Daniels, R. S., Douglas, A. R., Skehel, J. J., and Wiley, D. C. (1983) Analyses of the antigenicity of influenza haemagglutinin at the pH optimum for virus-mediated membrane fusion. J. Gen. Virol. 64, 1657–1662.CrossRefPubMedGoogle Scholar
  11. 11.
    Godley, L., Pfeifer, J., Steinhauer, D., et al. (1992) Introduction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity. Cell 68, 635–645.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Zhu-Nan Li
    • 1
  • David A. Steinhauer
    • 1
  1. 1.Emory University School of MedicineAtlanta

Personalised recommendations