Skip to main content
Biophysical Journal logoLink to Biophysical Journal
. 1999 Dec;77(6):3144–3151. doi: 10.1016/S0006-3495(99)77144-4

Hemifusion between cells expressing hemagglutinin of influenza virus and planar membranes can precede the formation of fusion pores that subsequently fully enlarge.

V I Razinkov 1, G B Melikyan 1, F S Cohen 1
PMCID: PMC1300584  PMID: 10585935

Abstract

The chronological relation between the establishment of lipid continuity and fusion pore formation has been investigated for fusion of cells expressing hemagglutinin (HA) of influenza virus to planar bilayer membranes. Self-quenching concentrations of lipid dye were placed in the planar membrane to monitor lipid mixing, and time-resolved admittance measurements were used to measure fusion pores. For rhodamine-PE, fusion pores always occurred before a detectable amount of dye moved into an HA-expressing cell. However, with DiI in the planar membrane, the relationship was reversed: the spread of dye preceded formation of small pores. In other words, by using DiI as probe, hemifusion was clearly observed to occur before pore formation. For hemifused cells, a small pore could form and subsequently fully enlarge. In contrast, for cells that express a glycosylphosphatidylinositol-anchored ectodomain of HA, hemifusion occurred, but no fully enlarged pores were observed. Therefore, the transmembrane domain of HA is required for the formation of fully enlarging pores. Thus, with the planar bilayer membranes as target, hemifusion can precede pore formation, and the occurrence of lipid dye spread does not preclude formation of pores that can enlarge fully.

Full Text

The Full Text of this article is available as a PDF (185.0 KB).

Selected References

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

  1. 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]
  2. 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]
  3. Chanturiya A., Chernomordik L. V., Zimmerberg J. Flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers. Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14423–14428. doi: 10.1073/pnas.94.26.14423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Chizmadzhev Y. A., Kumenko D. A., Kuzmin P. I., Chernomordik L. V., Zimmerberg J., Cohen F. S. Lipid flow through fusion pores connecting membranes of different tensions. Biophys J. 1999 Jun;76(6):2951–2965. doi: 10.1016/S0006-3495(99)77450-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Doxsey S. J., Sambrook J., Helenius A., White J. An efficient method for introducing macromolecules into living cells. J Cell Biol. 1985 Jul;101(1):19–27. doi: 10.1083/jcb.101.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Georgiou G. N., Morrison I. E., Cherry R. J. Digital fluorescence imaging of fusion of influenza virus with erythrocytes. FEBS Lett. 1989 Jul 3;250(2):487–492. doi: 10.1016/0014-5793(89)80782-3. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Kemble G. W., Danieli T., White J. M. Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion. Cell. 1994 Jan 28;76(2):383–391. doi: 10.1016/0092-8674(94)90344-1. [DOI] [PubMed] [Google Scholar]
  12. Kemble G. W., Henis Y. I., White J. M. GPI- and transmembrane-anchored influenza hemagglutinin differ in structure and receptor binding activity. J Cell Biol. 1993 Sep;122(6):1253–1265. doi: 10.1083/jcb.122.6.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lanzrein M., Käsermann N., Weingart R., Kempf C. Early events of Semliki Forest virus-induced cell-cell fusion. Virology. 1993 Oct;196(2):541–547. doi: 10.1006/viro.1993.1509. [DOI] [PubMed] [Google Scholar]
  14. Lee J., Lentz B. R. Evolution of lipidic structures during model membrane fusion and the relation of this process to cell membrane fusion. Biochemistry. 1997 May 27;36(21):6251–6259. doi: 10.1021/bi970404c. [DOI] [PubMed] [Google Scholar]
  15. Lindau M., Almers W. Structure and function of fusion pores in exocytosis and ectoplasmic membrane fusion. Curr Opin Cell Biol. 1995 Aug;7(4):509–517. doi: 10.1016/0955-0674(95)80007-7. [DOI] [PubMed] [Google Scholar]
  16. Lowy R. J., Sarkar D. P., Chen Y., Blumenthal R. Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1850–1854. doi: 10.1073/pnas.87.5.1850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Melikyan G. B., Deriy B. N., Ok D. C., Cohen F. S. Voltage-dependent translocation of R18 and DiI across lipid bilayers leads to fluorescence changes. Biophys J. 1996 Nov;71(5):2680–2691. doi: 10.1016/S0006-3495(96)79459-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Melikyan G. B., Niles W. D., Ratinov V. A., Karhanek M., Zimmerberg J., Cohen F. S. Comparison of transient and successful fusion pores connecting influenza hemagglutinin expressing cells to planar membranes. J Gen Physiol. 1995 Nov;106(5):803–819. doi: 10.1085/jgp.106.5.803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Melikyan G. B., White J. M., Cohen F. S. GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes. J Cell Biol. 1995 Nov;131(3):679–691. doi: 10.1083/jcb.131.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nanavati C., Markin V. S., Oberhauser A. F., Fernandez J. M. The exocytotic fusion pore modeled as a lipidic pore. Biophys J. 1992 Oct;63(4):1118–1132. doi: 10.1016/S0006-3495(92)81679-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Niles W. D., Cohen F. S. Fusion of influenza virions with a planar lipid membrane detected by video fluorescence microscopy. J Gen Physiol. 1991 Jun;97(6):1101–1119. doi: 10.1085/jgp.97.6.1101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nüssler F., Clague M. J., Herrmann A. Meta-stability of the hemifusion intermediate induced by glycosylphosphatidylinositol-anchored influenza hemagglutinin. Biophys J. 1997 Nov;73(5):2280–2291. doi: 10.1016/S0006-3495(97)78260-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pantazatos D. P., MacDonald R. C. Directly observed membrane fusion between oppositely charged phospholipid bilayers. J Membr Biol. 1999 Jul 1;170(1):27–38. doi: 10.1007/s002329900535. [DOI] [PubMed] [Google Scholar]
  25. Plonsky I., Zimmerberg J. The initial fusion pore induced by baculovirus GP64 is large and forms quickly. J Cell Biol. 1996 Dec;135(6 Pt 2):1831–1839. doi: 10.1083/jcb.135.6.1831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Qiao H., Armstrong R. T., Melikyan G. B., Cohen F. S., White J. M. A specific point mutant at position 1 of the influenza hemagglutinin fusion peptide displays a hemifusion phenotype. Mol Biol Cell. 1999 Aug;10(8):2759–2769. doi: 10.1091/mbc.10.8.2759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ratinov V., Plonsky I., Zimmerberg J. Fusion pore conductance: experimental approaches and theoretical algorithms. Biophys J. 1998 May;74(5):2374–2387. doi: 10.1016/S0006-3495(98)77946-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Razinkov V. I., Melikyan G. B., Epand R. M., Epand R. F., Cohen F. S. Effects of spontaneous bilayer curvature on influenza virus-mediated fusion pores. J Gen Physiol. 1998 Oct;112(4):409–422. doi: 10.1085/jgp.112.4.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Suzuki Y., Nagao Y., Kato H., Matsumoto M., Nerome K., Nakajima K., Nobusawa E. Human influenza A virus hemagglutinin distinguishes sialyloligosaccharides in membrane-associated gangliosides as its receptor which mediates the adsorption and fusion processes of virus infection. Specificity for oligosaccharides and sialic acids and the sequence to which sialic acid is attached. J Biol Chem. 1986 Dec 25;261(36):17057–17061. [PubMed] [Google Scholar]
  30. Tse F. W., Iwata A., Almers W. Membrane flux through the pore formed by a fusogenic viral envelope protein during cell fusion. J Cell Biol. 1993 May;121(3):543–552. doi: 10.1083/jcb.121.3.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wang H. Z., Veenstra R. D. Monovalent ion selectivity sequences of the rat connexin43 gap junction channel. J Gen Physiol. 1997 Apr;109(4):491–507. doi: 10.1085/jgp.109.4.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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