Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Dec 1;105(6):2887–2896. doi: 10.1083/jcb.105.6.2887

Anti-peptide antibodies detect steps in a protein conformational change: low-pH activation of the influenza virus hemagglutinin

PMCID: PMC2114698  PMID: 2447101

Abstract

At low pH, the hemagglutinin (HA) of influenza virus undergoes an irreversible conformational change that potentiates its essential membrane fusion function. We have probed the details of this conformational change using a panel of 14 anti-HA-peptide antibodies. Whereas some antibodies reacted equally well with both the neutral and low-pH HA conformations, others reacted to a significantly greater extent with the low-pH form. The locations of the peptides recognized by the latter antibodies in the three-dimensional HA structure indicated regions of the protein that change in response to low pH. Moreover, kinetic experiments suggested steps in the conformational change. In addition to their relevance to membrane fusion, our results show that anti-peptide antibodies can be used to study some types of biologically important protein conformational changes.

Full Text

The Full Text of this article is available as a PDF (3.8 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Balkema G. W., Dräger U. C. Light-dependent antibody labelling of photoreceptors. Nature. 1985 Aug 15;316(6029):630–633. doi: 10.1038/316630a0. [DOI] [PubMed] [Google Scholar]
  2. Both G. W., Sleigh M. J. Conservation and variation in the hemagglutinins of Hong Kong subtype influenza viruses during antigenic drift. J Virol. 1981 Sep;39(3):663–672. doi: 10.1128/jvi.39.3.663-672.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Campbell I. D., Dobson C. M., Williams R. J. The study of conformational states of proteins by nuclear magnetic resonance. Biochem J. 1985 Oct 1;231(1):1–10. doi: 10.1042/bj2310001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Connolly M. L. Solvent-accessible surfaces of proteins and nucleic acids. Science. 1983 Aug 19;221(4612):709–713. doi: 10.1126/science.6879170. [DOI] [PubMed] [Google Scholar]
  5. Daniels R. S., Douglas A. R., Skehel J. J., Wiley D. C. Analyses of the antigenicity of influenza haemagglutinin at the pH optimum for virus-mediated membrane fusion. J Gen Virol. 1983 Aug;64(Pt 8):1657–1662. doi: 10.1099/0022-1317-64-8-1657. [DOI] [PubMed] [Google Scholar]
  6. Daniels R. S., Downie J. C., Hay A. J., Knossow M., Skehel J. J., Wang M. L., Wiley D. C. Fusion mutants of the influenza virus hemagglutinin glycoprotein. Cell. 1985 Feb;40(2):431–439. doi: 10.1016/0092-8674(85)90157-6. [DOI] [PubMed] [Google Scholar]
  7. Doms R. W., Gething M. J., Henneberry J., White J., Helenius A. Variant influenza virus hemagglutinin that induces fusion at elevated pH. J Virol. 1986 Feb;57(2):603–613. doi: 10.1128/jvi.57.2.603-613.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doms R. W., Helenius A. Quaternary structure of influenza virus hemagglutinin after acid treatment. J Virol. 1986 Dec;60(3):833–839. doi: 10.1128/jvi.60.3.833-839.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Doms R. W., Helenius A., White J. Membrane fusion activity of the influenza virus hemagglutinin. The low pH-induced conformational change. J Biol Chem. 1985 Mar 10;260(5):2973–2981. [PubMed] [Google Scholar]
  10. Durham A. C., Finch J. T. Structure and roles of the polymorphic forms of tobacco mosaic virus protein. II. Electron microscope observations of the larger polymers. J Mol Biol. 1972 Jun 20;67(2):307–314. doi: 10.1016/0022-2836(72)90243-4. [DOI] [PubMed] [Google Scholar]
  11. Dyson H. J., Cross K. J., Houghten R. A., Wilson I. A., Wright P. E., Lerner R. A. The immunodominant site of a synthetic immunogen has a conformational preference in water for a type-II reverse turn. Nature. 1985 Dec 5;318(6045):480–483. doi: 10.1038/318480a0. [DOI] [PubMed] [Google Scholar]
  12. Gariépy J., Mietzner T. A., Schoolnik G. K. Peptide antisera as sequence-specific probes of protein conformational transitions: calmodulin exhibits calcium-dependent changes in antigenicity. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8888–8892. doi: 10.1073/pnas.83.23.8888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Geysen H. M., Tainer J. A., Rodda S. J., Mason T. J., Alexander H., Getzoff E. D., Lerner R. A. Chemistry of antibody binding to a protein. Science. 1987 Mar 6;235(4793):1184–1190. doi: 10.1126/science.3823878. [DOI] [PubMed] [Google Scholar]
  14. Green N., Alexander H., Olson A., Alexander S., Shinnick T. M., Sutcliffe J. G., Lerner R. A. Immunogenic structure of the influenza virus hemagglutinin. Cell. 1982 Mar;28(3):477–487. doi: 10.1016/0092-8674(82)90202-1. [DOI] [PubMed] [Google Scholar]
  15. Jackson D. C., Nestorowicz A. Antigenic determinants of influenza virus hemagglutinin. XI. Conformational changes detected by monoclonal antibodies. Virology. 1985 Aug;145(1):72–83. doi: 10.1016/0042-6822(85)90202-8. [DOI] [PubMed] [Google Scholar]
  16. Jou W. M., Verhoeyen M., Devos R., Saman E., Fang R., Huylebroeck D., Fiers W., Threlfall G., Barber C., Carey N. Complete structure of the hemagglutinin gene from the human influenza A/Victoria/3/75 (H3N2) strain as determined from cloned DNA. Cell. 1980 Mar;19(3):683–696. doi: 10.1016/s0092-8674(80)80045-6. [DOI] [PubMed] [Google Scholar]
  17. Kielian M., Helenius A. pH-induced alterations in the fusogenic spike protein of Semliki Forest virus. J Cell Biol. 1985 Dec;101(6):2284–2291. doi: 10.1083/jcb.101.6.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lear J. D., DeGrado W. F. Membrane binding and conformational properties of peptides representing the NH2 terminus of influenza HA-2. J Biol Chem. 1987 May 15;262(14):6500–6505. [PubMed] [Google Scholar]
  19. Maeda T., Ohnishi S. Activation of influenza virus by acidic media causes hemolysis and fusion of erythrocytes. FEBS Lett. 1980 Dec 29;122(2):283–287. doi: 10.1016/0014-5793(80)80457-1. [DOI] [PubMed] [Google Scholar]
  20. Millar J. R., Shaw P. J., Stammers D. K., Watson H. C. The low-resolution structure of human muscle aldolase. Philos Trans R Soc Lond B Biol Sci. 1981 Jun 26;293(1063):209–214. doi: 10.1098/rstb.1981.0074. [DOI] [PubMed] [Google Scholar]
  21. Niman H. L., Houghten R. A., Walker L. E., Reisfeld R. A., Wilson I. A., Hogle J. M., Lerner R. A. Generation of protein-reactive antibodies by short peptides is an event of high frequency: implications for the structural basis of immune recognition. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4949–4953. doi: 10.1073/pnas.80.16.4949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ruigrok R. W., Wrigley N. G., Calder L. J., Cusack S., Wharton S. A., Brown E. B., Skehel J. J. Electron microscopy of the low pH structure of influenza virus haemagglutinin. EMBO J. 1986 Jan;5(1):41–49. doi: 10.1002/j.1460-2075.1986.tb04175.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Skehel J. J., Bayley P. M., Brown E. B., Martin S. R., Waterfield M. D., White J. M., Wilson I. A., Wiley D. C. Changes in the conformation of influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion. Proc Natl Acad Sci U S A. 1982 Feb;79(4):968–972. doi: 10.1073/pnas.79.4.968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Skehel J. J., Stevens D. J., Daniels R. S., Douglas A. R., Knossow M., Wilson I. A., Wiley D. C. A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1779–1783. doi: 10.1073/pnas.81.6.1779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stegmann T., Hoekstra D., Scherphof G., Wilschut J. Fusion activity of influenza virus. A comparison between biological and artificial target membrane vesicles. J Biol Chem. 1986 Aug 25;261(24):10966–10969. [PubMed] [Google Scholar]
  26. Unwin P. N., Ennis P. D. Two configurations of a channel-forming membrane protein. Nature. 1984 Feb 16;307(5952):609–613. doi: 10.1038/307609a0. [DOI] [PubMed] [Google Scholar]
  27. Verhoeyen M., Fang R., Jou W. M., Devos R., Huylebroeck D., Saman E., Fiers W. Antigenic drift between the haemagglutinin of the Hong Kong influenza strains A/Aichi/2/68 and A/Victoria/3/75. Nature. 1980 Aug 21;286(5775):771–776. doi: 10.1038/286771a0. [DOI] [PubMed] [Google Scholar]
  28. Webster R. G., Brown L. E., Jackson D. C. Changes in the antigenicity of the hemagglutinin molecule of H3 influenza virus at acidic pH. Virology. 1983 Apr 30;126(2):587–599. doi: 10.1016/s0042-6822(83)80015-4. [DOI] [PubMed] [Google Scholar]
  29. White J., Helenius A., Gething M. J. Haemagglutinin of influenza virus expressed from a cloned gene promotes membrane fusion. Nature. 1982 Dec 16;300(5893):658–659. doi: 10.1038/300658a0. [DOI] [PubMed] [Google Scholar]
  30. White J., Kielian M., Helenius A. Membrane fusion proteins of enveloped animal viruses. Q Rev Biophys. 1983 May;16(2):151–195. doi: 10.1017/s0033583500005072. [DOI] [PubMed] [Google Scholar]
  31. Wiley D. C., Wilson I. A., Skehel J. J. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature. 1981 Jan 29;289(5796):373–378. doi: 10.1038/289373a0. [DOI] [PubMed] [Google Scholar]
  32. Wilson I. A., Niman H. L., Houghten R. A., Cherenson A. R., Connolly M. L., Lerner R. A. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. doi: 10.1016/0092-8674(84)90412-4. [DOI] [PubMed] [Google Scholar]
  33. Wilson I. A., Skehel J. J., Wiley D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981 Jan 29;289(5796):366–373. doi: 10.1038/289366a0. [DOI] [PubMed] [Google Scholar]
  34. Yewdell J. W., Gerhard W., Bachi T. Monoclonal anti-hemagglutinin antibodies detect irreversible antigenic alterations that coincide with the acid activation of influenza virus A/PR/834-mediated hemolysis. J Virol. 1983 Oct;48(1):239–248. doi: 10.1128/jvi.48.1.239-248.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zhao J. M., London E. Similarity of the conformation of diphtheria toxin at high temperature to that in the membrane-penetrating low-pH state. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2002–2006. doi: 10.1073/pnas.83.7.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES