Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1993 Feb;12(2):693–701. doi: 10.1002/j.1460-2075.1993.tb05703.x

Membrane fusion of Semliki Forest virus in a model system: correlation between fusion kinetics and structural changes in the envelope glycoprotein.

R Bron 1, J M Wahlberg 1, H Garoff 1, J Wilschut 1
PMCID: PMC413255  PMID: 8440260

Abstract

This paper presents a kinetic analysis of low-pH-induced fusion of Semliki Forest virus (SFV) with cholesterol-containing unilamellar lipid vesicles (liposomes), consisting otherwise of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin. Fusion is monitored continuously with a lipid mixing assay, involving virus bio-synthetically labeled with the fluorophore pyrene. At pH 5.55, 37 degrees C, SFV-liposome fusion occurs on the time scale of seconds. Extensive fusion (up to 60% of the virus) requires an excess of liposomes, while a low-pH preincubation of the virus alone results in inactivation of its fusion capacity. The onset of fusion after acidification of virus-liposome mixtures is preceded by a pH- and temperature-dependent lag phase. Early in this lag phase, a conformational change in the E2E1 spike glycoprotein occurs, involving formation of a trypsin-resistant E1 homotrimer, exposing a conformation-specific epitope (E1"). These changes are followed by a rapid, cholesterol-dependent binding of the virus to the liposomes (as assessed by sucrose density gradient analysis), subsequent fusion starting only after an additional delay. This sequence of events strongly suggests that the E1 homotrimeric structure represents the fusion-active conformation of the SFV spike, the actual fusion complex possibly involving a higher order oligomer of E1 trimers.

Full text

PDF
693

Images in this article

696Image
on p.696
698Image
on p.698

Selected References

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

  1. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  2. Boere W. A., Harmsen T., Vinjé J., Benaissa-Trouw B. J., Kraaijeveld C. A., Snippe H. Identification of distinct antigenic determinants on Semliki Forest virus by using monoclonal antibodies with different antiviral activities. J Virol. 1984 Nov;52(2):575–582. doi: 10.1128/jvi.52.2.575-582.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brunner J., Zugliani C., Mischler R. Fusion activity of influenza virus PR8/34 correlates with a temperature-induced conformational change within the hemagglutinin ectodomain detected by photochemical labeling. Biochemistry. 1991 Mar 5;30(9):2432–2438. doi: 10.1021/bi00223a019. [DOI] [PubMed] [Google Scholar]
  4. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  5. Clague M. J., Schoch C., Blumenthal R. Delay time for influenza virus hemagglutinin-induced membrane fusion depends on hemagglutinin surface density. J Virol. 1991 May;65(5):2402–2407. doi: 10.1128/jvi.65.5.2402-2407.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clague M. J., Schoch C., Zech L., Blumenthal R. Gating kinetics of pH-activated membrane fusion of vesicular stomatitis virus with cells: stopped-flow measurements by dequenching of octadecylrhodamine fluorescence. Biochemistry. 1990 Feb 6;29(5):1303–1308. doi: 10.1021/bi00457a028. [DOI] [PubMed] [Google Scholar]
  7. Cutler D. F., Garoff H. Mutants of the membrane-binding region of Semliki Forest virus E2 protein. I. Cell surface transport and fusogenic activity. J Cell Biol. 1986 Mar;102(3):889–901. doi: 10.1083/jcb.102.3.889. [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. Edwards J., Mann E., Brown D. T. Conformational changes in Sindbis virus envelope proteins accompanying exposure to low pH. J Virol. 1983 Mar;45(3):1090–1097. doi: 10.1128/jvi.45.3.1090-1097.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Flynn D. C., Meyer W. J., Mackenzie J. M., Jr, Johnston R. E. A conformational change in Sindbis virus glycoproteins E1 and E2 is detected at the plasma membrane as a consequence of early virus-cell interaction. J Virol. 1990 Aug;64(8):3643–3653. doi: 10.1128/jvi.64.8.3643-3653.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fries E., Helenius A. Binding of Semliki Forest virus and its spike glycoproteins to cells. Eur J Biochem. 1979 Jun;97(1):213–220. doi: 10.1111/j.1432-1033.1979.tb13105.x. [DOI] [PubMed] [Google Scholar]
  12. Galla H. J., Hartmann W. Excimer-forming lipids in membrane research. Chem Phys Lipids. 1980 Oct;27(3):199–219. doi: 10.1016/0009-3084(80)90036-5. [DOI] [PubMed] [Google Scholar]
  13. Galla H. J., Sackmann E. Lateral diffusion in the hydrophobic region of membranes: use of pyrene excimers as optical probes. Biochim Biophys Acta. 1974 Feb 26;339(1):103–115. doi: 10.1016/0005-2736(74)90336-8. [DOI] [PubMed] [Google Scholar]
  14. Garoff H., Frischauf A. M., Simons K., Lehrach H., Delius H. Nucleotide sequence of cdna coding for Semliki Forest virus membrane glycoproteins. Nature. 1980 Nov 20;288(5788):236–241. doi: 10.1038/288236a0. [DOI] [PubMed] [Google Scholar]
  15. Helenius A., Kartenbeck J., Simons K., Fries E. On the entry of Semliki forest virus into BHK-21 cells. J Cell Biol. 1980 Feb;84(2):404–420. doi: 10.1083/jcb.84.2.404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Helenius A. Semliki Forest virus penetration from endosomes: a morphological study. Biol Cell. 1984;51(2):181–185. doi: 10.1111/j.1768-322x.1984.tb00297.x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Kielian M. C., Helenius A. Role of cholesterol in fusion of Semliki Forest virus with membranes. J Virol. 1984 Oct;52(1):281–283. doi: 10.1128/jvi.52.1.281-283.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kielian M. C., Marsh M., Helenius A. Kinetics of endosome acidification detected by mutant and wild-type Semliki Forest virus. EMBO J. 1986 Dec 1;5(12):3103–3109. doi: 10.1002/j.1460-2075.1986.tb04616.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Kielian M., Jungerwirth S., Sayad K. U., DeCandido S. Biosynthesis, maturation, and acid activation of the Semliki Forest virus fusion protein. J Virol. 1990 Oct;64(10):4614–4624. doi: 10.1128/jvi.64.10.4614-4624.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Levy-Mintz P., Kielian M. Mutagenesis of the putative fusion domain of the Semliki Forest virus spike protein. J Virol. 1991 Aug;65(8):4292–4300. doi: 10.1128/jvi.65.8.4292-4300.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lobigs M., Wahlberg J. M., Garoff H. Spike protein oligomerization control of Semliki Forest virus fusion. J Virol. 1990 Oct;64(10):5214–5218. doi: 10.1128/jvi.64.10.5214-5218.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Marsh M., Bolzau E., Helenius A. Penetration of Semliki Forest virus from acidic prelysosomal vacuoles. Cell. 1983 Mar;32(3):931–940. doi: 10.1016/0092-8674(83)90078-8. [DOI] [PubMed] [Google Scholar]
  25. Marsh M., Wellsteed J., Kern H., Harms E., Helenius A. Monensin inhibits Semliki Forest virus penetration into culture cells. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5297–5301. doi: 10.1073/pnas.79.17.5297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mayer L. D., Hope M. J., Cullis P. R., Janoff A. S. Solute distributions and trapping efficiencies observed in freeze-thawed multilamellar vesicles. Biochim Biophys Acta. 1985 Jul 11;817(1):193–196. doi: 10.1016/0005-2736(85)90084-7. [DOI] [PubMed] [Google Scholar]
  27. Meyer W. J., Gidwitz S., Ayers V. K., Schoepp R. J., Johnston R. E. Conformational alteration of Sindbis virion glycoproteins induced by heat, reducing agents, or low pH. J Virol. 1992 Jun;66(6):3504–3513. doi: 10.1128/jvi.66.6.3504-3513.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Omar A., Koblet H. Semliki Forest virus particles containing only the E1 envelope glycoprotein are infectious and can induce cell-cell fusion. Virology. 1988 Sep;166(1):17–23. doi: 10.1016/0042-6822(88)90141-9. [DOI] [PubMed] [Google Scholar]
  29. Pal R., Barenholz Y., Wagner R. R. Pyrene phospholipid as a biological fluorescent probe for studying fusion of virus membrane with liposomes. Biochemistry. 1988 Jan 12;27(1):30–36. doi: 10.1021/bi00401a006. [DOI] [PubMed] [Google Scholar]
  30. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  31. Phalen T., Kielian M. Cholesterol is required for infection by Semliki Forest virus. J Cell Biol. 1991 Feb;112(4):615–623. doi: 10.1083/jcb.112.4.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Salminen A., Wahlberg J. M., Lobigs M., Liljeström P., Garoff H. Membrane fusion process of Semliki Forest virus. II: Cleavage-dependent reorganization of the spike protein complex controls virus entry. J Cell Biol. 1992 Jan;116(2):349–357. doi: 10.1083/jcb.116.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schmid S., Fuchs R., Kielian M., Helenius A., Mellman I. Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants. J Cell Biol. 1989 Apr;108(4):1291–1300. doi: 10.1083/jcb.108.4.1291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Stegmann T., Doms R. W., Helenius A. Protein-mediated membrane fusion. Annu Rev Biophys Biophys Chem. 1989;18:187–211. doi: 10.1146/annurev.bb.18.060189.001155. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. 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]
  41. 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]
  42. Wahlberg J. M., Boere W. A., Garoff H. The heterodimeric association between the membrane proteins of Semliki Forest virus changes its sensitivity to low pH during virus maturation. J Virol. 1989 Dec;63(12):4991–4997. doi: 10.1128/jvi.63.12.4991-4997.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wahlberg J. M., Bron R., Wilschut J., Garoff H. Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein. J Virol. 1992 Dec;66(12):7309–7318. doi: 10.1128/jvi.66.12.7309-7318.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wahlberg J. M., Garoff H. Membrane fusion process of Semliki Forest virus. I: Low pH-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells. J Cell Biol. 1992 Jan;116(2):339–348. doi: 10.1083/jcb.116.2.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. White J. M. Viral and cellular membrane fusion proteins. Annu Rev Physiol. 1990;52:675–697. doi: 10.1146/annurev.ph.52.030190.003331. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. White J., Helenius A. pH-dependent fusion between the Semliki Forest virus membrane and liposomes. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3273–3277. doi: 10.1073/pnas.77.6.3273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. White J., Kartenbeck J., Helenius A. Fusion of Semliki forest virus with the plasma membrane can be induced by low pH. J Cell Biol. 1980 Oct;87(1):264–272. doi: 10.1083/jcb.87.1.264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. White J., Matlin K., Helenius A. Cell fusion by Semliki Forest, influenza, and vesicular stomatitis viruses. J Cell Biol. 1981 Jun;89(3):674–679. doi: 10.1083/jcb.89.3.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wilschut J., Scholma J., Stegmann T. Molecular mechanisms of membrane fusion and applications of membrane fusion techniques. Adv Exp Med Biol. 1988;238:105–126. doi: 10.1007/978-1-4684-7908-9_9. [DOI] [PubMed] [Google Scholar]
  52. Wunderli-Allenspach H., Ott S. Kinetics of fusion and lipid transfer between virus receptor containing liposomes and influenza viruses as measured with the octadecylrhodamine B chloride assay. Biochemistry. 1990 Feb 27;29(8):1990–1997. doi: 10.1021/bi00460a005. [DOI] [PubMed] [Google Scholar]
  53. de Curtis I., Simons K. Dissection of Semliki Forest virus glycoprotein delivery from the trans-Golgi network to the cell surface in permeabilized BHK cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8052–8056. doi: 10.1073/pnas.85.21.8052. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES