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.

  • Letter
  • Published:

The hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes

Nature Microbiology volume 1, Article number: 16050 (2016) Cite this article

Subjects

Abstract

Influenza A virus haemagglutinin conformational change drives the membrane fusion of viral and endosomal membranes at low pH. Membrane fusion proceeds through an intermediate called hemifusion1,2. For viral fusion, the hemifusion structures are not determined3. Here, influenza virus-like particles4 carrying wild-type haemagglutinin or haemagglutinin hemifusion mutant G1S5 and liposome mixtures were studied at low pH by Volta phase plate cryo-electron tomography, which improves the signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm and a novel structure termed a ‘lipidic junction’. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by haemagglutinin. The rupture frequency and hemifusion diaphragm diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that haemagglutinin induces a merger between the viral and target membranes by one of two independent pathways: a rupture–insertion pathway leading to the lipidic junction and a hemifusion-stalk pathway leading to a fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch because of its negative spontaneous curvature in the target bilayer, as determined by continuum analysis.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: VLP structural features are resolved using VPP and indicate that influenza VLPs carrying G1S mutated HA engage one to seven HA glycoproteins on binding to liposomes.
Figure 2: Defocus–phase contrast cET and VPP–cET of liposomal membrane, Y-shaped lipidic junctions and hemifusion diaphragms.
Figure 3: HA spikes in close proximity to lipidic junctions and ruptured membranes.
Figure 4: Two independent, cholesterol concentration-dependent pathways lead to HDs of different diameter.

Similar content being viewed by others

References

  1. Chernomordik, L. V., Zimmerberg, J. & Kozlov, M. M. Membranes of the world unite! J. Cell Biol. 175, 201–207 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Harrison, S. C. Viral membrane fusion. Virology 479–480C, 498–507 (2015).

    Article  Google Scholar 

  4. Chen, B. J., Leser, G. P., Morita, E. & Lamb, R. A. Influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles. J. Virol. 81, 7111–7123 (2007).

    Article  CAS  Google Scholar 

  5. 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 10, 2759–2769 (1999).

    Article  CAS  Google Scholar 

  6. Chlanda, P. et al. Structural analysis of the roles of influenza A virus membrane-associated proteins in assembly and morphology. J. Virol. 89, 8957–8966 (2015).

    Article  CAS  Google Scholar 

  7. Harris, A. et al. Influenza virus pleiomorphy characterized by cryoelectron tomography. Proc. Natl Acad. Sci. USA 103, 19123–19127 (2006).

    Article  CAS  Google Scholar 

  8. Bonnafous, P. et al. Treatment of influenza virus with beta-propiolactone alters viral membrane fusion. Biochim. Biophys. Acta 1838, 355–363 (2014).

    Article  CAS  Google Scholar 

  9. Lee, K. K. Architecture of a nascent viral fusion pore. EMBO J. 29, 1299–1311 (2010).

    Article  CAS  Google Scholar 

  10. Maurer, U. E., Sodeik, B. & Grunewald, K. Native 3D intermediates of membrane fusion in herpes simplex virus 1 entry. Proc. Natl Acad. Sci. USA 105, 10559–10564 (2008).

    Article  CAS  Google Scholar 

  11. Erickson, H. P. & Klug, A. Measurement and compensation of defocusing and aberrations by Fourier processing of electron micrographs. Phil. Trans. R. Soc. Lond. B 261, 105–118 (1971).

    Article  Google Scholar 

  12. Danev, R., Buijsse, B., Khoshouei, M., Plitzko, J. M. & Baumeister, W. Volta potential phase plate for in-focus phase contrast transmission electron microscopy. Proc. Natl Acad. Sci. USA 111, 15635–15640 (2014).

    Article  CAS  Google Scholar 

  13. Kozlovsky, Y. & Kozlov, M. M. Stalk model of membrane fusion: solution of energy crisis. Biophys. J. 82, 882–895 (2002).

    Article  CAS  Google Scholar 

  14. Frolov, V. A., Dunina-Barkovskaya, A. Y., Samsonov, A. V. & Zimmerberg, J. Membrane permeability changes at early stages of influenza hemagglutinin-mediated fusion. Biophys. J. 85, 1725–1733 (2003).

    Article  CAS  Google Scholar 

  15. Chernomordik, L. V. & Kozlov, M. M. Mechanics of membrane fusion. Nature Struct. Mol. Biol. 15, 675–683 (2008).

    Article  CAS  Google Scholar 

  16. Ivanovic, T., Choi, J. L., Whelan, S. P., van Oijen, A. M. & Harrison, S. C. Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates. eLife 2, e00333 (2013).

    Article  Google Scholar 

  17. 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  Google Scholar 

  18. Kozlovsky, Y., Chernomordik, L. V. & Kozlov, M. M. Lipid intermediates in membrane fusion: formation, structure, and decay of hemifusion diaphragm. Biophys. J. 83, 2634–2651 (2002).

    Article  CAS  Google Scholar 

  19. Needham, D. & Nunn, R. S. Elastic deformation and failure of lipid bilayer membranes containing cholesterol. Biophys. J. 58, 997–1009 (1990).

    Article  CAS  Google Scholar 

  20. Zimmerberg, J. & Gawrisch, K. The physical chemistry of biological membranes. Nature Chem. Biol. 2, 564–567 (2006).

    Article  CAS  Google Scholar 

  21. Hao, M. et al. Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle. J. Biol. Chem. 277, 609–617 (2002).

    Article  CAS  Google Scholar 

  22. Van Meer, G., Voelker, D. R. & Feigenson, G. W. Membrane lipids: where they are and how they behave. Nature Rev. Mol. Cell Biol. 9, 112–124 (2008).

    Article  CAS  Google Scholar 

  23. Kobayashi, T. et al. Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nature Cell Biol. 1, 113–118 (1999).

    Article  CAS  Google Scholar 

  24. Yang, S. T., Zaitseva, E., Chernomordik, L. V. & Melikov, K. Cell-penetrating peptide induces leaky fusion of liposomes containing late endosome-specific anionic lipid. Biophys. J. 99, 2525–2533 (2010).

    Article  CAS  Google Scholar 

  25. Diao, J. et al. Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion. eLife 1, e00109 (2012).

    Article  Google Scholar 

  26. Kreutzberger, A. J., Kiessling, V. & Tamm, L. K. High cholesterol obviates a prolonged hemifusion intermediate in fast SNARE-mediated membrane fusion. Biophys. J. 109, 319–329 (2015).

    Article  CAS  Google Scholar 

  27. Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991).

    Article  CAS  Google Scholar 

  28. Ellens, H., Doxsey, S., Glenn, J. S. & White, J. M. Delivery of macromolecules into cells expressing a viral membrane fusion protein. Methods Cell Biol. 31, 155–178 (1989).

    Article  CAS  Google Scholar 

  29. Fukuda, Y., Laugks, U., Lucic, V., Baumeister, W. & Danev, R. Electron cryotomography of vitrified cells with a Volta phase plate. J. Struct. Biol. 190, 143–154 (2015).

    Article  Google Scholar 

  30. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  Google Scholar 

  31. Frangakis, A. S. & Hegerl, R. Noise reduction in electron tomographic reconstructions using nonlinear anisotropic diffusion. J. Struct. Biol. 135, 239–250 (2001).

    Article  CAS  Google Scholar 

  32. Helfrich, W. Elastic properties of lipid bilayers: theory and possible experiments. Z. Naturforsch C 28, 693–703 (1973).

    Article  CAS  Google Scholar 

  33. Nagle, J. F. & Tristram-Nagle, S. Structure of lipid bilayers. Biochim. Biophys. Acta 1469, 159–195 (2000).

    Article  CAS  Google Scholar 

  34. Hamm, M. & Kozlov, M. M. Elastic energy of tilt and bending of fluid membranes. Eur. Phys. J. 3, 323–335 (2000).

    CAS  Google Scholar 

  35. Siegel, D. P. & Kozlov, M. M. The Gaussian curvature elastic modulus of N-monomethylated dioleoylphosphatidylethanolamine: relevance to membrane fusion and lipid phase behavior. Biophys. J. 87, 366–374 (2004).

    Article  CAS  Google Scholar 

  36. Li, S. et al. pH-controlled two-step uncoating of influenza virus. Biophys. J. 106, 1447–1456 (2014).

    Article  CAS  Google Scholar 

  37. Pan, J., Tristram-Nagle, S. & Nagle, J. F. Effect of cholesterol on structural and mechanical properties of membranes depends on lipid chain saturation. Phys. Rev. E 80, 021931 (2009).

    Article  Google Scholar 

  38. Calder, L. J., Wasilewski, S., Berriman, J. A. & Rosenthal, P. B. Structural organization of a filamentous influenza A virus. Proc. Natl Acad. Sci. USA 107, 10685–10690 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank V. Nair for assistance with the Krios transmission electron microscope at Rocky Mountain Laboratories Microscopy Unit, National Institute of Allergy and Infectious Diseases, National Institutes of Health. The authors also thank L.-A. Carlson, L. Chernomordik, I. Morales and T. Reese for critical reading of the manuscript. This work was supported by the Division of Intramural Research of the Intramural Program of the National Institutes of Health.

Author information

Authors and Affiliations

  1. Section on Integrative Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, 20892, Maryland, USA

    Petr Chlanda, Elena Mekhedov, Hang Waters, Paul S. Blank & Joshua Zimmerberg

  2. Rocky Mountain Laboratories, Electron Microscopy Unit, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton 59840, Montana, USA

    Cindi L. Schwartz & Elizabeth R. Fischer

  3. Department of Mathematics, Fordham University, Bronx, 10458, New York, USA

    Rolf J. Ryham

  4. Department of Molecular Biophysics and Physiology, Rush University, Chicago, 60612, Illinois, USA

    Fredric S. Cohen

Authors
  1. Petr Chlanda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elena Mekhedov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hang Waters
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cindi L. Schwartz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Elizabeth R. Fischer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Rolf J. Ryham
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Fredric S. Cohen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Paul S. Blank
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Joshua Zimmerberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

The project was planned by P.C., P.S.B. and J.Z. Experimental work was performed by P.C. and E.M. pCAGGS-M1 and pCAGGS-M2 constructs were generated by H.W. Cryo-electron microscopy and tomography was done by P.C., C.L.S. and E.R.F. Statistical analysis and image processing was performed by P.C. and P.S.B. Continuum analysis was carried out by R.J.R. and F.S.C. The manuscript was written by P.C., R.J.R., F.S.C., P.S.B. and J.Z. All authors assisted in editing the manuscript and contributed to data analysis.

Corresponding authors

Correspondence to Petr Chlanda or Joshua Zimmerberg.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Results, Figures 1–10, Tables 1–4, Video legends and References. (PDF 14180 kb)

Supplementary Video 1

Tomogram showing ‘Y’ (lipidic) junctions mediated by G1S VLP corresponding to Fig. 2b. Tomogram was acquired at defocus –5 μm without VPP and denoised using nonlinear anisotropic diffusion (NAD) filter with k value 10 and 10 iterations. Scale bar: 50 nm. (MOV 2721 kb)

Supplementary Video 2

Tomogram showing ‘Y’ (lipidic) junctions mediated by G1S VLP corresponding to Fig. 2c. Tomogram was acquired at defocus –1 μm with VPP and denoised using NAD filter with k value 10 and 10 iterations. Scale bar: 50 nm. (MOV 6384 kb)

Supplementary Video 3

Tomogram showing liposome and complete HD connected to the membrane of the G1S filamentous VLP from top (corresponding to Fig. 2e). Tomogram was acquired at defocus –1 μm with VPP. (MOV 907 kb)

Supplementary Video 4

Animated visualization of the isosurface of a spherical VLP and several liposomes at low pH shown in Figs 2c and 3a–d. Liposome marked by A corresponds to the liposome in Figs (MOV 6651 kb)

Supplementary Video 5

Tomogram showing ruptured liposomes and lipidic junctions with G1S filamentous VLP (corresponding to Fig. 3e). Tomogram was acquired at defocus –1 μm with VPP and denoised using the NAD filter with a k value of 1 for 10 iterations. (MOV 2543 kb)

Supplementary Video 6

Animated visualization of the isosurface of ruptured liposomes and lipidic junctions with G1S filamentous VLP (corresponding to Fig. 3e). Scale bar: 50 nm. (MOV 2901 kb)

Supplementary Video 7

Tomogram showing WT VLP and liposomes fusion product (FP) after complete fusion characteristic of areas of membrane free of influenza glycoproteins. Red arrows and marks sparsely distributed influenza glycoproteins and red line highlights the membrane harbouring influenza glycoproteins. The unmarked area is free of influenza glycoproteins. Scale bar: 50 nm. (MOV 11311 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chlanda, P., Mekhedov, E., Waters, H. et al. The hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes. Nat Microbiol 1, 16050 (2016). https://doi.org/10.1038/nmicrobiol.2016.50

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nmicrobiol.2016.50

This article is cited by

Search

Advanced 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