Fusion of influenza virions with a planar lipid membrane detected by video fluorescence microscopy (original) (raw)

Abstract

The fusion of individual influenza virions with a planar phospholipid membrane was detected by fluorescence video microscopy. Virion envelopes were loaded with the lipophilic fluorescent marker octadecylrhodamine B (R18) to a density at which the fluorescence of the probe was self-quenched. Labeled virions were ejected toward the planar membrane from a micropipette in a custom-built video fluorescence microscope. Once a virion fused with the planar membrane, the marker was free to diffuse, and its fluorescence became dequenched, producing a flash of light. This flash was detected as a transient spot of light which increased and then diminished in brightness. The diffusion constants calculated from the brightness profiles for the flashes are consistent with fusion of virus to the membrane with consequent free diffusion of probe within the planar membrane. Under conditions known to be fusigenic for influenza virus (low pH and 37 degrees C), flashes appeared at a high rate and the planar membrane quickly became fluorescent. To further establish that these flashes were due to fusion, we showed that red blood cells, which normally do not attach to planar membranes, were able to bind to membranes that had been exposed to virus under fusigenic conditions. The amount of binding correlated with the amount of flashing. This indicates that flashes signaled the reconstitution of the hemagglutinin glycoprotein (HA) of influenza virus, a well-known erythrocyte receptor, into the planar membrane, as would be expected in a fusion process. The flash rate on ganglioside-containing asolectin membranes increased as the pH was lowered. This is also consistent with the known fusion behavior of influenza virus with cell membranes and with phospholipid vesicles. We conclude that the flashes result from the fusion of individual virions to the planar membrane.

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Selected References

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  1. Akabas M. H., Cohen F. S., Finkelstein A. Separation of the osmotically driven fusion event from vesicle-planar membrane attachment in a model system for exocytosis. J Cell Biol. 1984 Mar;98(3):1063–1071. doi: 10.1083/jcb.98.3.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Caton A. J., Brownlee G. G., Yewdell J. W., Gerhard W. The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell. 1982 Dec;31(2 Pt 1):417–427. doi: 10.1016/0092-8674(82)90135-0. [DOI] [PubMed] [Google Scholar]
  3. Cohen F. S., Zimmerberg J., Finkelstein A. Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane. J Gen Physiol. 1980 Mar;75(3):251–270. doi: 10.1085/jgp.75.3.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Fahey P. F., Webb W. W. Lateral diffusion in phospholipid bilayer membranes and multilamellar liquid crystals. Biochemistry. 1978 Jul 25;17(15):3046–3053. doi: 10.1021/bi00608a016. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Harter C., Bächi T., Semenza G., Brunner J. Hydrophobic photolabeling identifies BHA2 as the subunit mediating the interaction of bromelain-solubilized influenza virus hemagglutinin with liposomes at low pH. Biochemistry. 1988 Mar 22;27(6):1856–1864. doi: 10.1021/bi00406a010. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Huang R. T., Rott R., Klenk H. D. Influenza viruses cause hemolysis and fusion of cells. Virology. 1981 Apr 15;110(1):243–247. doi: 10.1016/0042-6822(81)90030-1. [DOI] [PubMed] [Google Scholar]
  11. Johnston P. A., Jahn R., Südhof T. C. Transmembrane topography and evolutionary conservation of synaptophysin. J Biol Chem. 1989 Jan 15;264(2):1268–1273. [PubMed] [Google Scholar]
  12. Karlsson K. A. Animal glycosphingolipids as membrane attachment sites for bacteria. Annu Rev Biochem. 1989;58:309–350. doi: 10.1146/annurev.bi.58.070189.001521. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Montal M., Mueller P. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3561–3566. doi: 10.1073/pnas.69.12.3561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Morris S. J., Sarkar D. P., White J. M., Blumenthal R. Kinetics of pH-dependent fusion between 3T3 fibroblasts expressing influenza hemagglutinin and red blood cells. Measurement by dequenching of fluorescence. J Biol Chem. 1989 Mar 5;264(7):3972–3978. [PubMed] [Google Scholar]
  16. Niles W. D., Cohen F. S. The role of N-acetylneuraminic (sialic) acid in the pH dependence of influenza virion fusion with planar phospholipid membranes. J Gen Physiol. 1991 Jun;97(6):1121–1140. doi: 10.1085/jgp.97.6.1121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Niles W. D., Cohen F. S. Video fluorescence microscopy studies of phospholipid vesicle fusion with a planar phospholipid membrane. Nature of membrane-membrane interactions and detection of release of contents. J Gen Physiol. 1987 Nov;90(5):703–735. doi: 10.1085/jgp.90.5.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Niles W. D., Levis R. A., Cohen F. S. Planar bilayer membranes made from phospholipid monolayers form by a thinning process. Biophys J. 1988 Mar;53(3):327–335. doi: 10.1016/S0006-3495(88)83110-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Perin M. S., Fried V. A., Mignery G. A., Jahn R., Südhof T. C. Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature. 1990 May 17;345(6272):260–263. doi: 10.1038/345260a0. [DOI] [PubMed] [Google Scholar]
  20. Perrin D., Langley O. K., Aunis D. Anti-alpha-fodrin inhibits secretion from permeabilized chromaffin cells. Nature. 1987 Apr 2;326(6112):498–501. doi: 10.1038/326498a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Sarkar D. P., Morris S. J., Eidelman O., Zimmerberg J., Blumenthal R. Initial stages of influenza hemagglutinin-induced cell fusion monitored simultaneously by two fluorescent events: cytoplasmic continuity and lipid mixing. J Cell Biol. 1989 Jul;109(1):113–122. doi: 10.1083/jcb.109.1.113. [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. 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]
  25. Stegmann T., Hoekstra D., Scherphof G., Wilschut J. Kinetics of pH-dependent fusion between influenza virus and liposomes. Biochemistry. 1985 Jun 18;24(13):3107–3113. doi: 10.1021/bi00334a006. [DOI] [PubMed] [Google Scholar]
  26. Trimble W. S., Cowan D. M., Scheller R. H. VAMP-1: a synaptic vesicle-associated integral membrane protein. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4538–4542. doi: 10.1073/pnas.85.12.4538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weis W., Brown J. H., Cusack S., Paulson J. C., Skehel J. J., Wiley D. C. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988 Jun 2;333(6172):426–431. doi: 10.1038/333426a0. [DOI] [PubMed] [Google Scholar]
  28. Wharton S. A., Skehel J. J., Wiley D. C. Studies of influenza haemagglutinin-mediated membrane fusion. Virology. 1986 Feb;149(1):27–35. doi: 10.1016/0042-6822(86)90083-8. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  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., 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]
  33. Zimmerberg J., Cohen F. S., Finkelstein A. Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. I. Discharge of vesicular contents across the planar membrane. J Gen Physiol. 1980 Mar;75(3):241–250. doi: 10.1085/jgp.75.3.241. [DOI] [PMC free article] [PubMed] [Google Scholar]