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.

Progressive ordering with decreasing temperature of the phospholipids of influenza virus

Abstract

Using linewidth and spinning sideband intensities of lipid hydrocarbon chain resonances in proton magic angle spinning NMR spectra, we detected the temperature-dependent phase state of naturally occurring lipids of intact influenza virus without exogenous probes. Increasingly, below 41 °C ordered and disordered lipid domains coexisted for the viral envelope and extracts thereof. At 22 °C much lipid was in a gel phase, the fraction of which reversibly increased with cholesterol depletion. Diffusion measurements and fluorescence microscopy independently confirmed the existence of gel-phase domains. Thus the existence of ordered regions of lipids in biological membranes is now demonstrated. Above the physiological temperatures of influenza infection, the physical properties of viral envelope lipids, regardless of protein content, were indistinguishable from those of the disordered fraction. Viral fusion appears to be uncorrelated to ordered lipid content. Lipid ordering may contribute to viral stability at lower temperatures, which has recently been found to be critical for airborne transmission.

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: 1H MAS NMR spectra of model and biomembranes.
Figure 2: 1H MAS NMR spectra as a function of temperature.
Figure 3: Fraction of ordered lipids as a function of temperature.
Figure 4: 1H MAS NMR diffusion measurements.
Figure 5: Fluorescence microscopy detection of ordered lipid domains.
Figure 6: Effect of temperature on lipid mixing assay of influenza virus fusion with RBC membranes.

References

  1. Lenard, J. & Compans, R.W. The membrane structure of lipid-containing viruses. Biochim. Biophys. Acta 344, 51–94 (1974).

    Article  CAS  Google Scholar 

  2. Pessin, J.E. & Glaser, M. Budding of Rous sarcoma virus and vesicular stomatitis virus from localized lipid regions in the plasma membrane of chicken embryo fibroblasts. J. Biol. Chem. 255, 9044–9050 (1980).

    CAS  PubMed  Google Scholar 

  3. van Meer, G. & Simons, K. Viruses budding from either the apical or the basolateral plasma-membrane domain of Mdck cells have unique phospholipid compositions. EMBO J. 1, 847–852 (1982).

    Article  CAS  Google Scholar 

  4. Blom, T.S. et al. Mass spectrometric analysis reveals an increase in plasma membrane polyunsaturated phospholipid species upon cellular cholesterol loading. Biochemistry 40, 14635–14644 (2001).

    Article  CAS  Google Scholar 

  5. Ipsen, J.H., Karlstrom, G., Mouritsen, O.G., Wennerstrom, H. & Zuckermann, M.J. Phase equilibria in the phosphatidylcholine-cholesterol system. Biochim. Biophys. Acta 905, 162–172 (1987).

    Article  CAS  Google Scholar 

  6. Brown, D.A. & London, E. Structure and origin of ordered lipid domains in biological membranes. J. Membr. Biol. 164, 103–114 (1998).

    Article  CAS  Google Scholar 

  7. Simons, K. & Ikonen, E. Functional rafts in cell membranes. Nature 387, 569–572 (1997).

    Article  CAS  Google Scholar 

  8. Lichtenberg, D., Goni, F.M. & Heerklotz, H. Detergent-resistant membranes should not be identified with membrane rafts. Trends Biochem. Sci. 30, 430–436 (2005).

    Article  CAS  Google Scholar 

  9. Jacobson, K., Mouritsen, O.G. & Anderson, R.G.W. Lipid rafts: at a crossroad between cell biology and physics. Nat. Cell Biol. 9, 7–14 (2007).

    Article  CAS  Google Scholar 

  10. Sefton, B.M. & Gaffney, B.J. Effect of viral proteins on fluidity of membrane lipids in Sindbis virus. J. Mol. Biol. 90, 343–358 (1974).

    Article  CAS  Google Scholar 

  11. Landsberger, F.R. & Compans, R.W. Effect of membrane-protein on lipid bilayer structure - spin-label electron-spin resonance study of vesicular stomatitis-virus. Biochemistry 15, 2356–2360 (1976).

    Article  CAS  Google Scholar 

  12. Landsberger, F.R., Lenard, J., Paxton, J. & Compans, R.W. Spin-label electron spin resonance study of lipid-containing membrane of influenza virus. Proc. Natl. Acad. Sci. USA 68, 2579–2583 (1971).

    Article  CAS  Google Scholar 

  13. Scheiffele, P., Rietveld, A., Wilk, T. & Simons, K. Influenza viruses select ordered lipid domains during budding from the plasma membrane. J. Biol. Chem. 274, 2038–2044 (1999).

    Article  CAS  Google Scholar 

  14. Bukrinskaya, A.G., Molotkovsky, J.G., Vodovozova, E.L., Manevich, Y.M. & Bergelson, L.D. The molecular-organization of the influenza-virus surface - studies using photoreactive and fluorescent labeled phospholipid probes. Biochim. Biophys. Acta 897, 285–292 (1987).

    Article  CAS  Google Scholar 

  15. Lenard, J. Virus envelopes and plasma-membranes. Annu. Rev. Biophys. Bioeng. 7, 139–165 (1978).

    Article  CAS  Google Scholar 

  16. Hess, S.T. et al. Quantitative electron microscopy and fluorescence spectroscopy of the membrane distribution of influenza hemagglutinin. J. Cell Biol. 169, 965–976 (2005).

    Article  CAS  Google Scholar 

  17. Dietrich, C. et al. Lipid rafts reconstituted in model membranes. Biophys. J. 80, 1417–1428 (2001).

    Article  CAS  Google Scholar 

  18. Veatch, S.L. & Keller, S.L. Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol. Biophys. J. 85, 3074–3083 (2003).

    Article  CAS  Google Scholar 

  19. Baumgart, T., Hess, S.T. & Webb, W.W. Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension. Nature 425, 821–824 (2003).

    Article  CAS  Google Scholar 

  20. Polozov, I.V. & Gawrisch, K. Domains in binary SOPC/POPE lipid mixtures studied by pulsed field gradient 1H MAS NMR. Biophys. J. 87, 1741–1751 (2004).

    Article  CAS  Google Scholar 

  21. Polozov, I.V. & Gawrisch, K. Characterization of the liquid-ordered state by proton MAS NMR. Biophys. J. 90, 2051–2061 (2006).

    Article  CAS  Google Scholar 

  22. Veatch, S.L., Polozov, I.V., Gawrisch, K. & Keller, S.L. Liquid domains in vesicles investigated by NMR and fluorescence microscopy. Biophys. J. 86, 2910–2922 (2004).

    Article  CAS  Google Scholar 

  23. Oldfield, E., Bowers, J.L. & Forbes, J. High-resolution proton and carbon-13 NMR of membranes: why sonicate? Biochemistry 26, 6919–6923 (1987).

    Article  CAS  Google Scholar 

  24. Huster, D., Arnold, K. & Gawrisch, K. Influence of docosahexaenoic acid and cholesterol on lateral lipid organization in phospholipid mixtures. Biochemistry 37, 17299–17308 (1998).

    Article  CAS  Google Scholar 

  25. Gaede, H.C. & Gawrisch, K. Lateral diffusion rates of lipid, water, and a hydrophobic drug in a multilamellar liposome. Biophys. J. 85, 1734–1740 (2003).

    Article  CAS  Google Scholar 

  26. Rothman, J.E., Tsai, D.K., Dawidowicz, E.A. & Lenard, J. Transbilayer phospholipid asymmetry and its maintenance in membrane of influenza-virus. Biochemistry 15, 2361–2370 (1976).

    Article  CAS  Google Scholar 

  27. Stegmann, T. et al. Functional reconstitution of influenza-virus envelopes. EMBO J. 6, 2651–2659 (1987).

    Article  CAS  Google Scholar 

  28. Filippov, A., Oradd, G. & Lindblom, G. The effect of cholesterol on the lateral diffusion of phospholipids in oriented bilayers. Biophys. J. 84, 3079–3086 (2003).

    Article  CAS  Google Scholar 

  29. Scheidt, H.A., Huster, D. & Gawrisch, K. Diffusion of cholesterol and its precursors in lipid membranes studied by 1H PFG MAS NMR. Biophys. J. 89, 2504–2512 (2005).

    Article  CAS  Google Scholar 

  30. Hac, A.E., Seeger, H.M., Fidorra, M. & Heimburg, T. Diffusion in two-component lipid membranes—a fluorescence correlation spectroscopy and Monte Carlo simulation study. Biophys. J. 88, 317–333 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  32. Hess, S.T. et al. Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories. Proc. Natl. Acad. Sci. USA 104, 17370–17375 (2007).

    Article  CAS  Google Scholar 

  33. Filippov, A., Oradd, G. & Lindblom, G. Sphingomyelin structure influences the lateral diffusion and raft formation in lipid bilayers. Biophys. J. 90, 2086–2092 (2006).

    Article  CAS  Google Scholar 

  34. Koynova, R. & Caffrey, M. Phases and phase-transitions of the sphingolipids. Biochim. Biophys. Acta 1255, 213–236 (1995).

    Article  Google Scholar 

  35. Clarke, J.A., Heron, A.J., Seddon, J.M. & Law, R.V. The diversity of the liquid ordered (L-o) phase of phosphatidylcholine/cholesterol membranes: a variable temperature multinuclear solid-state NMR and X-ray diffraction study. Biophys. J. 90, 2383–2393 (2006).

    Article  CAS  Google Scholar 

  36. McMullen, T.P. & McElhaney, R.N. New aspects of the interaction of cholesterol with dipalmitoylphosphatidylcholine bilayers as revealed by high-sensitivity differential scanning calorimetry. Biochim. Biophys. Acta 1234, 90–98 (1995).

    Article  Google Scholar 

  37. Polozov, I.V., Molotkovsky, J.G. & Bergelson, L.D. Anthrylvinyl-labeled phospholipids as membrane probes - the phosphatidylcholine-phosphatidylethanolamine system. Chem. Phys. Lipids 69, 209–218 (1994).

    Article  CAS  Google Scholar 

  38. Heberle, F.A., Buboltz, J.T., Stringer, D. & Feigenson, G.W. Fluorescence methods to detect phase boundaries in lipid bilayer mixtures. Biochim. Biophys. Acta 1746, 186–192 (2005).

    Article  CAS  Google Scholar 

  39. Kuzmin, P.I., Akimov, S.A., Chizmadzhev, Y.A., Zimmerberg, J. & Cohen, F.S. Line tension and interaction energies of membrane rafts calculated from lipid splay and tilt. Biophys. J. 88, 1120–1133 (2005).

    Article  CAS  Google Scholar 

  40. Sun, X. & Whittaker, G.R. Role for influenza virus envelope cholesterol in virus entry and infection. J. Virol. 77, 12543–12551 (2003).

    Article  CAS  Google Scholar 

  41. Takeda, M., Leser, G.P., Russell, C.J. & Lamb, R.A. Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion. Proc. Natl. Acad. Sci. USA 100, 14610–14617 (2003).

    Article  CAS  Google Scholar 

  42. Campbell, S. et al. The raft-promoting property of virion-associated cholesterol, but not the presence of virion-associated Brij 98 rafts, is a determinant of human immunodeficiency virus type 1 infectivity. J. Virol. 78, 10556–10565 (2004).

    Article  CAS  Google Scholar 

  43. Silvius, J.R. Role of cholesterol in lipid raft formation: lessons from lipid model systems. Biochim. Biophys. Acta 1610, 174–183 (2003).

    Article  CAS  Google Scholar 

  44. Niu, S.L. & Litman, B.J. Determination of membrane cholesterol partition coefficient using a lipid vesicle-cyclodextrin binary system: effect of phospholipid acyl chain unsaturation and headgroup composition. Biophys. J. 83, 3408–3415 (2002).

    Article  CAS  Google Scholar 

  45. Lowen, A.C., Mubareka, S., Steel, J. & Palese, P. Influenza virus transmission is dependent on relative humidity and temperature. PLoS Pathog. 3, 1470–1476 (2007).

    Article  CAS  Google Scholar 

  46. Paterson, R.G. & Lamb, R.A. Molecular Virology: a Practical Approach (eds. Davidson, A. & Elliott, R.M.) 35–73 (IRL Oxford University Press, Oxford, 1993).

    Google Scholar 

  47. Hoekstra, D., Deboer, T., Klappe, K. & Wilschut, J. Fluorescence method for measuring the kinetics of fusion between biological membranes. Biochemistry 23, 5675–5681 (1984).

    Article  CAS  Google Scholar 

  48. Klein, U., Gimpl, G. & Fahrenholz, F. Alteration of the myometrial plasma-membrane cholesterol content with beta-cyclodextrin modulates the binding-affinity of the oxytocin receptor. Biochemistry 34, 13784–13793 (1995).

    Article  CAS  Google Scholar 

  49. Folch, J., Lees, M. & Sloane-Stanley, G.H. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509 (1957).

    CAS  PubMed  Google Scholar 

  50. Metsikko, K., Vanmeer, G. & Simons, K. Reconstitution of the fusogenic activity of vesicular stomatitis-virus. EMBO J. 5, 3429–3435 (1986).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by funding from the intramural research programs of the National Institute of Child Health and Human Development and the National Institute on Alcohol Abuse and Alcoholism, US National Institutes of Health. We thank L. Chernomordik and E. Leikina for conducting the experiments on content mixing.

Author information

Authors and Affiliations

Authors

Contributions

I.V.P., K.G. and J.Z. designed research, analyzed data and wrote the paper. I.V.P. and L.B. performed research.

Corresponding author

Correspondence to Joshua Zimmerberg.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1 and 2, Supplementary Discussion and Supplementary Methods (PDF 507 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Polozov, I., Bezrukov, L., Gawrisch, K. et al. Progressive ordering with decreasing temperature of the phospholipids of influenza virus. Nat Chem Biol 4, 248–255 (2008). https://doi.org/10.1038/nchembio.77

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.77

This article is cited by

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