Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1998 Jan;74(1):54–62. doi: 10.1016/S0006-3495(98)77766-5

Morphological changes and fusogenic activity of influenza virus hemagglutinin.

T Shangguan 1, D P Siegel 1, J D Lear 1, P H Axelsen 1, D Alford 1, J Bentz 1
PMCID: PMC1299361  PMID: 9449309

Abstract

The kinetics of low-pH induced fusion of influenza virus with liposomes have been compared to changes in the morphology of influenza hemagglutinin (HA). At pH 4.9 and 30 degrees C, the fusion of influenza A/PR/8/34 virus with ganglioside-bearing liposomes was complete within 6 min. Virus preincubated at pH 4.9 and 30 degrees C in the absence of liposomes for 2 or 10 min retained most of its fusion activity. However, fusion activity was dramatically reduced after 30 min, and virtually abolished after a 60-min preincubation. Cryo-electron microscopy showed that the hemagglutinin spikes of virions exposed to pH 4.9 at 30 degrees C for 10 min underwent no major morphological changes. After 30 min, however, the spike morphology changed dramatically, and further changes occurred for up to 60 min after exposure to low pH. Because the morphological changes occur at a rate corresponding to the loss of fusion activity, and because these changes are much slower than the rate at which fusion occurs, we conclude that the morphologically altered HA is inactive with respect to fusion-promoting activity. Molecular modeling studies indicate that the formation of an extended coiled coil within the HA trimer, as proposed for HA at low pH, requires a major conformational change in HA, and that the morphological changes we observe are consistent with the formation of an extended coiled coil. These results imply that the crystallographically determined low-pH form of HA does occur in the intact virus, but that this form is not a precursor of viral fusion. It is speculated that the motion to the low-pH form may be responsible for the membrane destabilization leading to fusion.

Full Text

The Full Text of this article is available as a PDF (1,008.4 KB).

Selected References

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

  1. Alford D., Ellens H., Bentz J. Fusion of influenza virus with sialic acid-bearing target membranes. Biochemistry. 1994 Mar 1;33(8):1977–1987. doi: 10.1021/bi00174a002. [DOI] [PubMed] [Google Scholar]
  2. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  3. Bellare J. R., Davis H. T., Scriven L. E., Talmon Y. Controlled environment vitrification system: an improved sample preparation technique. J Electron Microsc Tech. 1988 Sep;10(1):87–111. doi: 10.1002/jemt.1060100111. [DOI] [PubMed] [Google Scholar]
  4. Bentz J., Ellens H., Alford D. An architecture for the fusion site of influenza hemagglutinin. FEBS Lett. 1990 Dec 10;276(1-2):1–5. doi: 10.1016/0014-5793(90)80492-2. [DOI] [PubMed] [Google Scholar]
  5. Bentz J. Intermediates and kinetics of membrane fusion. Biophys J. 1992 Aug;63(2):448–459. doi: 10.1016/S0006-3495(92)81622-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Booy F. P., Ruigrok R. W., van Bruggen E. F. Electron microscopy of influenza virus. A comparison of negatively stained and ice-embedded particles. J Mol Biol. 1985 Aug 20;184(4):667–676. doi: 10.1016/0022-2836(85)90312-2. [DOI] [PubMed] [Google Scholar]
  7. Brand C. M., Skehel J. J. Crystalline antigen from the influenza virus envelope. Nat New Biol. 1972 Aug 2;238(83):145–147. doi: 10.1038/newbio238145a0. [DOI] [PubMed] [Google Scholar]
  8. Bullough P. A., Hughson F. M., Skehel J. J., Wiley D. C. Structure of influenza haemagglutinin at the pH of membrane fusion. Nature. 1994 Sep 1;371(6492):37–43. doi: 10.1038/371037a0. [DOI] [PubMed] [Google Scholar]
  9. Carr C. M., Kim P. S. A spring-loaded mechanism for the conformational change of influenza hemagglutinin. Cell. 1993 May 21;73(4):823–832. doi: 10.1016/0092-8674(93)90260-w. [DOI] [PubMed] [Google Scholar]
  10. Carr C. M., Kim P. S. Flu virus invasion: halfway there. Science. 1994 Oct 14;266(5183):234–236. doi: 10.1126/science.7939658. [DOI] [PubMed] [Google Scholar]
  11. Chan D. C., Fass D., Berger J. M., Kim P. S. Core structure of gp41 from the HIV envelope glycoprotein. Cell. 1997 Apr 18;89(2):263–273. doi: 10.1016/s0092-8674(00)80205-6. [DOI] [PubMed] [Google Scholar]
  12. Chen J., Wharton S. A., Weissenhorn W., Calder L. J., Hughson F. M., Skehel J. J., Wiley D. C. A soluble domain of the membrane-anchoring chain of influenza virus hemagglutinin (HA2) folds in Escherichia coli into the low-pH-induced conformation. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12205–12209. doi: 10.1073/pnas.92.26.12205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Ellens H., Bentz J., Mason D., Zhang F., White J. M. Fusion of influenza hemagglutinin-expressing fibroblasts with glycophorin-bearing liposomes: role of hemagglutinin surface density. Biochemistry. 1990 Oct 16;29(41):9697–9707. doi: 10.1021/bi00493a027. [DOI] [PubMed] [Google Scholar]
  15. Fujiyoshi Y., Kume N. P., Sakata K., Sato S. B. Fine structure of influenza A virus observed by electron cryo-microscopy. EMBO J. 1994 Jan 15;13(2):318–326. doi: 10.1002/j.1460-2075.1994.tb06264.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gething M. J., Doms R. W., York D., White J. Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus. J Cell Biol. 1986 Jan;102(1):11–23. doi: 10.1083/jcb.102.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Godley L., Pfeifer J., Steinhauer D., Ely B., Shaw G., Kaufmann R., Suchanek E., Pabo C., Skehel J. J., Wiley D. C. Introduction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity. Cell. 1992 Feb 21;68(4):635–645. doi: 10.1016/0092-8674(92)90140-8. [DOI] [PubMed] [Google Scholar]
  18. Günther-Ausborn S., Praetor A., Stegmann T. Inhibition of influenza-induced membrane fusion by lysophosphatidylcholine. J Biol Chem. 1995 Dec 8;270(49):29279–29285. doi: 10.1074/jbc.270.49.29279. [DOI] [PubMed] [Google Scholar]
  19. Hernandez L. D., Hoffman L. R., Wolfsberg T. G., White J. M. Virus-cell and cell-cell fusion. Annu Rev Cell Dev Biol. 1996;12:627–661. doi: 10.1146/annurev.cellbio.12.1.627. [DOI] [PubMed] [Google Scholar]
  20. Korte T., Ludwig K., Krumbiegel M., Zirwer D., Damaschun G., Herrmann A. Transient changes of the conformation of hemagglutinin of influenza virus at low pH detected by time-resolved circular dichroism spectroscopy. J Biol Chem. 1997 Apr 11;272(15):9764–9770. doi: 10.1074/jbc.272.15.9764. [DOI] [PubMed] [Google Scholar]
  21. Puri A., Booy F. P., Doms R. W., White J. M., Blumenthal R. Conformational changes and fusion activity of influenza virus hemagglutinin of the H2 and H3 subtypes: effects of acid pretreatment. J Virol. 1990 Aug;64(8):3824–3832. doi: 10.1128/jvi.64.8.3824-3832.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ruigrok R. W., Aitken A., Calder L. J., Martin S. R., Skehel J. J., Wharton S. A., Weis W., Wiley D. C. Studies on the structure of the influenza virus haemagglutinin at the pH of membrane fusion. J Gen Virol. 1988 Nov;69(Pt 11):2785–2795. doi: 10.1099/0022-1317-69-11-2785. [DOI] [PubMed] [Google Scholar]
  23. Ruigrok R. W., Hewat E. A., Wade R. H. Low pH deforms the influenza virus envelope. J Gen Virol. 1992 Apr;73(Pt 4):995–998. doi: 10.1099/0022-1317-73-4-995. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Sato S. B., Kawasaki K., Ohnishi S. Hemolytic activity of influenza virus hemagglutinin glycoproteins activated in mildly acidic environments. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3153–3157. doi: 10.1073/pnas.80.11.3153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shangguan T., Alford D., Bentz J. Influenza-virus-liposome lipid mixing is leaky and largely insensitive to the material properties of the target membrane. Biochemistry. 1996 Apr 16;35(15):4956–4965. doi: 10.1021/bi9526903. [DOI] [PubMed] [Google Scholar]
  27. Siegel D. P. Energetics of intermediates in membrane fusion: comparison of stalk and inverted micellar intermediate mechanisms. Biophys J. 1993 Nov;65(5):2124–2140. doi: 10.1016/S0006-3495(93)81256-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Siegel D. P., Green W. J., Talmon Y. The mechanism of lamellar-to-inverted hexagonal phase transitions: a study using temperature-jump cryo-electron microscopy. Biophys J. 1994 Feb;66(2 Pt 1):402–414. doi: 10.1016/s0006-3495(94)80790-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Silvius J. R., Leventis R., Brown P. M., Zuckermann M. Novel fluorescent phospholipids for assays of lipid mixing between membranes. Biochemistry. 1987 Jul 14;26(14):4279–4287. doi: 10.1021/bi00388a015. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Stegmann T., Booy F. P., Wilschut J. Effects of low pH on influenza virus. Activation and inactivation of the membrane fusion capacity of the hemagglutinin. J Biol Chem. 1987 Dec 25;262(36):17744–17749. [PubMed] [Google Scholar]
  32. Stegmann T., Delfino J. M., Richards F. M., Helenius A. The HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion. J Biol Chem. 1991 Sep 25;266(27):18404–18410. [PubMed] [Google Scholar]
  33. Stegmann T., Nir S., Wilschut J. Membrane fusion activity of influenza virus. Effects of gangliosides and negatively charged phospholipids in target liposomes. Biochemistry. 1989 Feb 21;28(4):1698–1704. doi: 10.1021/bi00430a041. [DOI] [PubMed] [Google Scholar]
  34. Stegmann T., White J. M., Helenius A. Intermediates in influenza induced membrane fusion. EMBO J. 1990 Dec;9(13):4231–4241. doi: 10.1002/j.1460-2075.1990.tb07871.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Steinhauer D. A., Martín J., Lin Y. P., Wharton S. A., Oldstone M. B., Skehel J. J., Wiley D. C. Studies using double mutants of the conformational transitions in influenza hemagglutinin required for its membrane fusion activity. Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12873–12878. doi: 10.1073/pnas.93.23.12873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Szoka F., Jr, Papahadjopoulos D. Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4194–4198. doi: 10.1073/pnas.75.9.4194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Weber T., Paesold G., Galli C., Mischler R., Semenza G., Brunner J. Evidence for H(+)-induced insertion of influenza hemagglutinin HA2 N-terminal segment into viral membrane. J Biol Chem. 1994 Jul 15;269(28):18353–18358. [PubMed] [Google Scholar]
  38. Weis W. I., Cusack S. C., Brown J. H., Daniels R. S., Skehel J. J., Wiley D. C. The structure of a membrane fusion mutant of the influenza virus haemagglutinin. EMBO J. 1990 Jan;9(1):17–24. doi: 10.1002/j.1460-2075.1990.tb08075.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wharton S. A., Calder L. J., Ruigrok R. W., Skehel J. J., Steinhauer D. A., Wiley D. C. Electron microscopy of antibody complexes of influenza virus haemagglutinin in the fusion pH conformation. EMBO J. 1995 Jan 16;14(2):240–246. doi: 10.1002/j.1460-2075.1995.tb06997.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. White J. M., Wilson I. A. Anti-peptide antibodies detect steps in a protein conformational change: low-pH activation of the influenza virus hemagglutinin. J Cell Biol. 1987 Dec;105(6 Pt 2):2887–2896. doi: 10.1083/jcb.105.6.2887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. White J., Kartenbeck J., Helenius A. Membrane fusion activity of influenza virus. EMBO J. 1982;1(2):217–222. doi: 10.1002/j.1460-2075.1982.tb01150.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wild C., Dubay J. W., Greenwell T., Baird T., Jr, Oas T. G., McDanal C., Hunter E., Matthews T. Propensity for a leucine zipper-like domain of human immunodeficiency virus type 1 gp41 to form oligomers correlates with a role in virus-induced fusion rather than assembly of the glycoprotein complex. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12676–12680. doi: 10.1073/pnas.91.26.12676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wiley D. C., Skehel J. J. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu Rev Biochem. 1987;56:365–394. doi: 10.1146/annurev.bi.56.070187.002053. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Yu Y. G., King D. S., Shin Y. K. Insertion of a coiled-coil peptide from influenza virus hemagglutinin into membranes. Science. 1994 Oct 14;266(5183):274–276. doi: 10.1126/science.7939662. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES