Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Conformational change and protein–protein interactions of the fusion protein of Semliki Forest virus

Abstract

Fusion of biological membranes is mediated by specific lipid-interacting proteins that induce the formation and expansion of an initial fusion pore. Here we report the crystal structure of the ectodomain of the Semliki Forest virus fusion glycoprotein E1 in its low-pH-induced trimeric form. E1 adopts a folded-back conformation that, in the final post-fusion form of the full-length protein, would bring the fusion peptide loop and the transmembrane anchor to the same end of a stable protein rod. The observed conformation of the fusion peptide loop is compatible with interactions only with the outer leaflet of the lipid bilayer. Crystal contacts between fusion peptide loops of adjacent E1 trimers, together with electron microscopy observations, suggest that in an early step of membrane fusion, an intermediate assembly of five trimers creates two opposing nipple-like deformations in the viral and target membranes, leading to formation of the fusion pore.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Overall fold of glycoprotein E1.
Figure 2: Trimer–trimer interactions observed in the 2D lattice and in the 3D crystals.
Figure 3: A ring of five trimers of E1*.
Figure 4: Model for membrane fusion involving protein–protein interactions, as explained in the text.

Similar content being viewed by others

References

  1. Jahn, R., Lang, T. & Sudhof, T. C. Membrane fusion. Cell 112, 519–533 (2003)

    Article  CAS  PubMed  Google Scholar 

  2. Skehel, J. J. & Wiley, D. C. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu. Rev. Biochem. 69, 531–569 (2000)

    Article  CAS  PubMed  Google Scholar 

  3. Weissenhorn, W. et al. Structural basis for membrane fusion by enveloped viruses. Mol. Membr. Biol. 16, 3–9 (1999)

    Article  CAS  PubMed  Google Scholar 

  4. 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. 133, 559–569 (1996)

    Article  CAS  PubMed  Google Scholar 

  5. 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. 135, 63–71 (1996)

    Article  CAS  PubMed  Google Scholar 

  6. Markovic, I., Leikina, E., Zhukovsky, M., Zimmerberg, J. & Chernomordik, L. V. Synchronized activation and refolding of influenza hemagglutinin in multimeric fusion machines. J. Cell Biol. 155, 833–844 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zimmerberg, J. & Chernomordik, L. V. Membrane fusion. Adv. Drug Deliv. Rev. 38, 197–205 (1999)

    Article  CAS  PubMed  Google Scholar 

  8. Blumenthal, R., Clague, M. J., Durell, S. R. & Epand, R. M. Membrane fusion. Chem. Rev. 103, 53–69 (2003)

    Article  CAS  PubMed  Google Scholar 

  9. Helenius, A., Kartenbeck, J., Simons, K. & Fries, E. On the entry of Semliki Forest virus into BHK-21 cells. J. Cell Biol. 84, 404–420 (1980)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Schlesinger, S. & Schlesinger, M. J. in Fields Virology (eds Knipe, D. M. & Howley, P. M.) 895–916 (Lippincott Williams and Wilkins, Philadelphia, 2001)

    Google Scholar 

  11. Lescar, J. et al. The fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal pH. Cell 105, 137–148 (2001)

    Article  CAS  PubMed  Google Scholar 

  12. Zhang, W. et al. Placement of the structural proteins in Sindbis virus. J. Virol. 76, 11645–11658 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Rey, F. A., Heinz, F. X., Mandl, C., Kunz, C. & Harrison, S. C. The envelope glycoprotein from tick-borne encephalitis virus at 2Å resolution. Nature 375, 291–298 (1995)

    Article  CAS  ADS  PubMed  Google Scholar 

  14. Modis, Y., Ogata, S., Clements, D. & Harrison, S. C. A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc. Natl Acad. Sci. USA 100, 6899–6901 (2003)

    Article  Google Scholar 

  15. 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. 116, 339–348 (1992)

    Article  CAS  PubMed  Google Scholar 

  16. Kielian, M., Chatterjee, P. K., Gibbons, D. L. & Lu, Y. E. in Subcellular Biochemistry Vol. 34 Fusion of Biological Membranes and Related Problems (eds Hilderson, H. & Fuller, S.) 409–455 (Plenum, New York, 2000)

    Google Scholar 

  17. Klimjack, M. R., Jeffrey, S. & Kielian, M. Membrane and protein interactions of a soluble form of the Semliki Forest virus fusion protein. J. Virol. 68, 6940–6946 (1994)

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Gibbons, D. L. & Kielian, M. Molecular dissection of the Semliki Forest virus homotrimer reveals two functionally distinct regions of the fusion protein. J. Virol. 76, 1194–1205 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ahn, A., Gibbons, D. L. & Kielian, M. The fusion peptide of Semliki Forest virus associates with sterol-rich membrane domains. J. Virol. 76, 3267–3275 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Gibbons, D. L. et al. Visualization of the target-membrane-inserted fusion protein of Semliki Forest virus by combined electron microscopy and crystallography. Cell 114, 573–583 (2003)

    Article  CAS  PubMed  Google Scholar 

  21. Eckert, D. M. & Kim, P. S. Mechanisms of viral membrane fusion and its inhibition. Annu. Rev. Biochem. 70, 777–810 (2001)

    Article  CAS  PubMed  Google Scholar 

  22. Wahlberg, J. M., Bron, R., Wilschut, J. & Garoff, H. Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein. J. Virol. 66, 7309–7318 (1992)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Markosyan, R. M., Cohen, F. S. & Melikyan, G. B. HIV-1 envelope proteins complete their folding into six-helix bundles immediately after fusion pore formation. Mol. Biol. Cell 14, 926–938 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kemble, G. W., Danieli, T. & White, J. M. Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion. Cell 76, 383–391 (1994)

    Article  CAS  PubMed  Google Scholar 

  25. Armstrong, R. T., Kushnir, A. S. & White, J. M. The transmembrane domain of influenza hemagglutinin exhibits a stringent length requirement to support the hemifusion to fusion transition. J. Cell Biol. 151, 425–437 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bagai, S. & Lamb, R. A. Truncation of the COOH-terminal region of the paramyxovirus SV5 fusion protein leads to hemifusion but not complete fusion. J. Cell Biol. 135, 73–84 (1996)

    Article  CAS  PubMed  Google Scholar 

  27. Januszeski, M. M., Cannon, P. M., Chen, D., Rozenberg, Y. & Anderson, W. F. Functional analysis of the cytoplasmic tail of Moloney murine leukemia virus envelope protein. J. Virol. 71, 3613–3619 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Melikyan, G. B., Markosyan, R. M., Brener, S. A., Rozenberg, Y. & Cohen, F. S. Role of the cytoplasmic tail of ecotropic moloney murine leukemia virus Env protein in fusion pore formation. J. Virol. 74, 447–455 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chernomordik, L., 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. 140, 1369–1382 (1998)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Gaudin, Y. Rabies virus-induced membrane fusion pathway. J. Cell Biol. 150, 601–612 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 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. 80, 812–821 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Modis, Y., Ogata, S., Clements, D. & Harrison, S. C. Structure of the dengue virus envelope protein after membrane fusion. Nature 427, 313–319 (2004)

    Article  CAS  ADS  PubMed  Google Scholar 

  33. Bressanelli, S. et al. Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. EMBO J. (in the press)

  34. Gibbons, D. L. et al. Purification and crystallization reveal two types of interactions of the fusion protein homotrimer of Semliki Forest virus. J. Virol. (in the press)

  35. Otwinowski, Z. & Minor, W. in Macromolecular Crystallography Part A (eds Carter, C. W. & Sweet, R. M.) 307–326 (Academic Press, London, 1997)

    Book  Google Scholar 

  36. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. de la Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–493 (1997)

    Article  CAS  PubMed  Google Scholar 

  38. Cowtan, K. dm: an automated procedure for phase improvement by density modification. Joint CCP4 ESF-EACBM Newslett. Protein Crystallogr. 31, 34–38 (1994)

    Google Scholar 

  39. Jones, T. A. & Kjeldgaard, M. in Macromolecular Crystallography Part B (eds Carter, C. W. & Sweet, R. M.) 173–208 (Academic Press, London, 1997)

    Book  Google Scholar 

  40. Roussel, A. & Cambillaud, C. Silicon Graphics Geometry Partners Directory (Silicon Graphics, Mountain View, California, 1991)

    Google Scholar 

  41. Brünger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  PubMed  Google Scholar 

  42. Carson, M. Ribbon models of macromolecules. J. Mol. Graph. 5, 103–106 (1987)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Bressanelli, S. Duquerroy and P. Fernandez Varela for their help at different stages of this work; A. Ahn and A. Urian for help with virus and protein preparation; C. Schulze-Briese and T. Tomikazi for help during diffraction data collection; Y. Gaudin for critically reading the manuscript; and J. Navaza for helpful discussions. More than 80% of the data used to determine the crystal structure were collected at synchrotron beam line X06SA of the Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland. Other synchrotron sources used were beam lines ID14 and ID29 at the European Synchrotron Radiation Facility, Grenoble, France, and beam line BW7A at DESY, Hamburg, Germany. M.K. acknowledges support from the Public Health Service and from a Cancer Center Core Support Grant from the National Cancer Institute. F.A.R. acknowledges support from the CNRS and INRA, the SESAME Program of the Région Ile-de-France, the French Fondation pour la Recherche Médicale, the Association pour la Recherche contre le Cancer, the CNRS programs “Physique et Chimie du Vivant” and “Dynamique et réactivité des assemblages biologiques”, and the European Union ENhcV consortium. D.L.G. was supported by the Medical Scientist Training Program of the Albert Einstein College of Medicine, the Albert Cass Traveling Fellowship and the CNRS.Authors' contributions The crystallographic analyses reported in this paper were performed by F.A.R. and M.C.V.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Margaret Kielian or Félix A. Rey.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gibbons, D., Vaney, MC., Roussel, A. et al. Conformational change and protein–protein interactions of the fusion protein of Semliki Forest virus. Nature 427, 320–325 (2004). https://doi.org/10.1038/nature02239

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature02239

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing