Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2001 Sep;81(3):1521–1535. doi: 10.1016/S0006-3495(01)75806-7

Comprehensive kinetic analysis of influenza hemagglutinin-mediated membrane fusion: role of sialate binding.

A Mittal 1, J Bentz 1
PMCID: PMC1301630  PMID: 11509365

Abstract

The data of Danieli et al. (J. Cell Biol. 133:559-569, 1996) and Blumenthal et al. (J. Cell Biol. 135:63-71, 1996) for fusion between hemagglutinin (HA)-expressing cells and fluorescently labeled erythrocytes has been analyzed using a recently published comprehensive mass action kinetic model for HA-mediated fusion. This model includes the measurable steps in the fusion process, i.e., first pore formation, lipid mixing, and content mixing of aqueous fluorescent markers. It contains two core parameters of the fusion site architecture. The first is the minimum number of aggregated HAs needed to sustain subsequent fusion intermediates. The second is the minimal number of those HAs within the fusogenic aggregate that must undergo a slow "essential" conformational change needed to initiate bilayer destabilization. Because the kinetic model has several parameters, each data set was exhaustively fitted to obtain all best fits. Although each of the data sets required particular parameter ranges for best fits, a consensus subset of these parameter ranges could fit all of the data. Thus, this comprehensive model subsumes the available mass action kinetic data for the fusion of HA-expressing cells with erythrocytes, despite the differences in assays and experimental design, which necessitated transforming fluorescence dequenching intensities to equivalent cumulative waiting time distributions. We find that HAs bound to sialates on glycophorin can participate in fusion as members of the fusogenic aggregate, but they cannot undergo the essential conformational change that initiates bilayer destabilization, thus solving a long-standing debate. Also, the similarity in rate constants for lipid mixing and content mixing found here for HA-mediated fusion and by Lee and Lentz (Proc. Natl. Acad. Sci. U.S.A. 95:9274-9279, 1998) for PEG-induced fusion of phosphatidylcholine liposomes supports the idea that subsequent to stable fusion pore formation, the evolution of fusion intermediates is determined more by the lipids than by the proteins.

Full Text

The Full Text of this article is available as a PDF (253.4 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. 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]
  3. 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]
  4. Bentz J. Membrane fusion mediated by coiled coils: a hypothesis. Biophys J. 2000 Feb;78(2):886–900. doi: 10.1016/S0006-3495(00)76646-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bentz J. Minimal aggregate size and minimal fusion unit for the first fusion pore of influenza hemagglutinin-mediated membrane fusion. Biophys J. 2000 Jan;78(1):227–245. doi: 10.1016/S0006-3495(00)76587-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bentz J., Mittal A. Deployment of membrane fusion protein domains during fusion. Cell Biol Int. 2000;24(11):819–838. doi: 10.1006/cbir.2000.0632. [DOI] [PubMed] [Google Scholar]
  7. Blumenthal R., Sarkar D. P., Durell S., Howard D. E., Morris S. J. Dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events. J Cell Biol. 1996 Oct;135(1):63–71. doi: 10.1083/jcb.135.1.63. [DOI] [PMC free article] [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. Böttcher C., Ludwig K., Herrmann A., van Heel M., Stark H. Structure of influenza haemagglutinin at neutral and at fusogenic pH by electron cryo-microscopy. FEBS Lett. 1999 Dec 17;463(3):255–259. doi: 10.1016/s0014-5793(99)01475-1. [DOI] [PubMed] [Google Scholar]
  10. Carr C. M., Chaudhry C., Kim P. S. Influenza hemagglutinin is spring-loaded by a metastable native conformation. Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14306–14313. doi: 10.1073/pnas.94.26.14306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Chen Y. D., Blumenthal R. On the use of self-quenching fluorophores in the study of membrane fusion kinetics. The effect of slow probe redistribution. Biophys Chem. 1989 Nov;34(3):283–292. doi: 10.1016/0301-4622(89)80065-1. [DOI] [PubMed] [Google Scholar]
  13. Chernomordik L. V., Frolov V. A., Leikina E., Bronk P., Zimmerberg J. The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation. J Cell Biol. 1998 Mar 23;140(6):1369–1382. doi: 10.1083/jcb.140.6.1369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chernomordik L. V., Leikina E., Frolov V., Bronk P., Zimmerberg J. An early stage of membrane fusion mediated by the low pH conformation of influenza hemagglutinin depends upon membrane lipids. J Cell Biol. 1997 Jan 13;136(1):81–93. doi: 10.1083/jcb.136.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Danieli T., Pelletier S. L., Henis Y. I., White J. M. Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers. J Cell Biol. 1996 May;133(3):559–569. doi: 10.1083/jcb.133.3.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Günther-Ausborn S., Schoen P., Bartoldus I., Wilschut J., Stegmann T. Role of hemagglutinin surface density in the initial stages of influenza virus fusion: lack of evidence for cooperativity. J Virol. 2000 Mar;74(6):2714–2720. doi: 10.1128/jvi.74.6.2714-2720.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Haque M. E., McIntosh T. J., Lentz B. R. Influence of lipid composition on physical properties and peg-mediated fusion of curved and uncurved model membrane vesicles: "nature's own" fusogenic lipid bilayer. Biochemistry. 2001 Apr 10;40(14):4340–4348. doi: 10.1021/bi002030k. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Hoekstra D., de Boer T., Klappe K., Wilschut J. Fluorescence method for measuring the kinetics of fusion between biological membranes. Biochemistry. 1984 Nov 20;23(24):5675–5681. doi: 10.1021/bi00319a002. [DOI] [PubMed] [Google Scholar]
  22. Kozlov M. M., Chernomordik L. V. A mechanism of protein-mediated fusion: coupling between refolding of the influenza hemagglutinin and lipid rearrangements. Biophys J. 1998 Sep;75(3):1384–1396. doi: 10.1016/S0006-3495(98)74056-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee J., Lentz B. R. Secretory and viral fusion may share mechanistic events with fusion between curved lipid bilayers. Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9274–9279. doi: 10.1073/pnas.95.16.9274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leikina E., Markovic I., Chernomordik L. V., Kozlov M. M. Delay of influenza hemagglutinin refolding into a fusion-competent conformation by receptor binding: a hypothesis. Biophys J. 2000 Sep;79(3):1415–1427. doi: 10.1016/S0006-3495(00)76393-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lentz B. R., Malinin V., Haque M. E., Evans K. Protein machines and lipid assemblies: current views of cell membrane fusion. Curr Opin Struct Biol. 2000 Oct;10(5):607–615. doi: 10.1016/s0959-440x(00)00138-x. [DOI] [PubMed] [Google Scholar]
  26. Marchesi V. T., Tillack T. W., Jackson R. L., Segrest J. P., Scott R. E. Chemical characterization and surface orientation of the major glycoprotein of the human erythrocyte membrane. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1445–1449. doi: 10.1073/pnas.69.6.1445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Markosyan R. M., Cohen F. S., Melikyan G. B. The lipid-anchored ectodomain of influenza virus hemagglutinin (GPI-HA) is capable of inducing nonenlarging fusion pores. Mol Biol Cell. 2000 Apr;11(4):1143–1152. doi: 10.1091/mbc.11.4.1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Markosyan R. M., Melikyan G. B., Cohen F. S. Evolution of intermediates of influenza virus hemagglutinin-mediated fusion revealed by kinetic measurements of pore formation. Biophys J. 2001 Feb;80(2):812–821. doi: 10.1016/S0006-3495(01)76060-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Melikyan G. B., Jin H., Lamb R. A., Cohen F. S. The role of the cytoplasmic tail region of influenza virus hemagglutinin in formation and growth of fusion pores. Virology. 1997 Aug 18;235(1):118–128. doi: 10.1006/viro.1997.8686. [DOI] [PubMed] [Google Scholar]
  30. Melikyan G. B., Lin S., Roth M. G., Cohen F. S. Amino acid sequence requirements of the transmembrane and cytoplasmic domains of influenza virus hemagglutinin for viable membrane fusion. Mol Biol Cell. 1999 Jun;10(6):1821–1836. doi: 10.1091/mbc.10.6.1821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Melikyan G. B., Markosyan R. M., Roth M. G., Cohen F. S. A point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion. Mol Biol Cell. 2000 Nov;11(11):3765–3775. doi: 10.1091/mbc.11.11.3765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Melikyan G. B., Niles W. D., Cohen F. S. The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface. J Gen Physiol. 1995 Nov;106(5):783–802. doi: 10.1085/jgp.106.5.783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Millar B. M., Calder L. J., Skehel J. J., Wiley D. C. Membrane fusion by surrogate receptor-bound influenza haemagglutinin. Virology. 1999 May 10;257(2):415–423. doi: 10.1006/viro.1999.9624. [DOI] [PubMed] [Google Scholar]
  34. Niles W. D., Cohen F. S. Single event recording shows that docking onto receptor alters the kinetics of membrane fusion mediated by influenza hemagglutinin. Biophys J. 1993 Jul;65(1):171–176. doi: 10.1016/S0006-3495(93)81049-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ohuchi M., Ohuchi R., Matsumoto A. Control of biological activities of influenza virus hemagglutinin by its carbohydrate moiety. Microbiol Immunol. 1999;43(12):1071–1076. doi: 10.1111/j.1348-0421.1999.tb03363.x. [DOI] [PubMed] [Google Scholar]
  36. Pisano A., Redmond J. W., Williams K. L., Gooley A. A. Glycosylation sites identified by solid-phase Edman degradation: O-linked glycosylation motifs on human glycophorin A. Glycobiology. 1993 Oct;3(5):429–435. doi: 10.1093/glycob/3.5.429. [DOI] [PubMed] [Google Scholar]
  37. Qiao H., Pelletier S. L., Hoffman L., Hacker J., Armstrong R. T., White J. M. Specific single or double proline substitutions in the "spring-loaded" coiled-coil region of the influenza hemagglutinin impair or abolish membrane fusion activity. J Cell Biol. 1998 Jun 15;141(6):1335–1347. doi: 10.1083/jcb.141.6.1335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sauter N. K., Bednarski M. D., Wurzburg B. A., Hanson J. E., Whitesides G. M., Skehel J. J., Wiley D. C. Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500-MHz proton nuclear magnetic resonance study. Biochemistry. 1989 Oct 17;28(21):8388–8396. doi: 10.1021/bi00447a018. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Shangguan T., Siegel D. P., Lear J. D., Axelsen P. H., Alford D., Bentz J. Morphological changes and fusogenic activity of influenza virus hemagglutinin. Biophys J. 1998 Jan;74(1):54–62. doi: 10.1016/S0006-3495(98)77766-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Skehel J. J., Wiley D. C. Coiled coils in both intracellular vesicle and viral membrane fusion. Cell. 1998 Dec 23;95(7):871–874. doi: 10.1016/s0092-8674(00)81710-9. [DOI] [PubMed] [Google Scholar]
  42. Stegmann T., Bartoldus I., Zumbrunn J. Influenza hemagglutinin-mediated membrane fusion: influence of receptor binding on the lag phase preceding fusion. Biochemistry. 1995 Feb 14;34(6):1825–1832. doi: 10.1021/bi00006a002. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. 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]
  46. Wolber P. K., Hudson B. S. An analytic solution to the Förster energy transfer problem in two dimensions. Biophys J. 1979 Nov;28(2):197–210. doi: 10.1016/S0006-3495(79)85171-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Zimmerberg J., Blumenthal R., Sarkar D. P., Curran M., Morris S. J. Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion. J Cell Biol. 1994 Dec;127(6 Pt 2):1885–1894. doi: 10.1083/jcb.127.6.1885. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES