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:

Carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins

Nature Immunology volume 6, pages 995–1001 (2005)Cite this article

Abstract

Defensins are peptides that protect the host against microorganisms. Here we show that the θ-defensin retrocyclin 2 (RC2) inhibited influenza virus infection by blocking membrane fusion mediated by the viral hemagglutinin. RC2 was effective even after hemagglutinin attained a fusogenic conformation or had induced membrane hemifusion. RC2, a multivalent lectin, prevented hemagglutinin-mediated fusion by erecting a network of crosslinked and immobilized surface glycoproteins. RC2 also inhibited fusion mediated by Sindbis virus and baculovirus. Human β-defensin 3 and mannan-binding lectin also blocked viral fusion by creating a protective barricade of immobilized surface proteins. This general mechanism might explain the broad-spectrum antiviral activity of many multivalent lectins of the innate immune system.

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: RC2 inhibits influenza virus entry into MDCK cells.
Figure 2: RC2 inhibits HA-mediated membrane fusion.
Figure 3: RC2 inhibits fusion mediated by baculovirus gp64 and Sindbis E1.
Figure 4: HA-mediated fusion involves cell surface carbohydrates.
Figure 5: RC2 immobilizes membrane proteins.
Figure 6: MBL and HBD3 inhibit fusion and immobilize membrane proteins.

Similar content being viewed by others

Accession codes

Accessions

BINDPlus

References

  1. Smith, A.E. & Helenius, A. How viruses enter animal cells. Science 304, 237–242 (2004).

    Article  CAS  Google Scholar 

  2. Lehrer, R.I. Primate defensins. Nat. Rev. Microbiol. 2, 727–738 (2004).

    Article  CAS  Google Scholar 

  3. Selsted, M.E. & Ouellette, A.J. Mammalian defensins in the antimicrobial immune response. Nat. Immunol. 6, 551–557 (2005).

    Article  CAS  Google Scholar 

  4. Owen, S.M. et al. RC-101, a retrocyclin-1 analogue with enhanced activity against primary HIV type 1 isolates. AIDS Res. Hum. Retroviruses 20, 1157–1165 (2004).

    Article  CAS  Google Scholar 

  5. Munk, C. et al. The θ-defensin, retrocyclin, inhibits HIV-1 entry. AIDS Res. Hum. Retroviruses 19, 875–881 (2003).

    Article  Google Scholar 

  6. Yasin, B. et al. Theta defensins protect cells from infection by herpes simplex virus by inhibiting viral adhesion and entry. J. Virol. 78, 5147–5156 (2004).

    Article  CAS  Google Scholar 

  7. Holmskov, U., Thiel, S. & Jensenius, J.C. Collections and ficolins: humoral lectins of the innate immune defense. Annu. Rev. Immunol. 21, 547–578 (2003).

    Article  CAS  Google Scholar 

  8. Etchison, J., Doyle, M., Penhoet, E. & Holland, J. Synthesis and cleavage of influenza virus proteins. J. Virol. 7, 155–167 (1971).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 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 

  10. 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  Google Scholar 

  11. Chernomordik, L.V., Leikina, E., Frolov, V., Bronk, P. & Zimmerberg, J. An early stage of membrane fusion mediated by the low pH conformation of influenza hemagglutinin depends upon membrane lipids. J. Cell Biol. 136, 81–94 (1997).

    Article  CAS  Google Scholar 

  12. Rust, M.J., Lakadamyali, M., Zhang, F. & Zhuang, X. Assembly of endocytic machinery around individual influenza viruses during viral entry. Nat. Struct. Mol. Biol. 11, 567–573 (2004).

    Article  CAS  Google Scholar 

  13. Wang, W. et al. Activity of α- and θ-defensins against primary isolates of HIV-1. J. Immunol. 173, 515–520 (2004).

    Article  CAS  Google Scholar 

  14. Schoch, C., Blumenthal, R. & Clague, M.J. A long-lived state for influenza virus-erythrocyte complexes committed to fusion at neutral pH. FEBS Lett. 311, 221–225 (1992).

    Article  CAS  Google Scholar 

  15. Korte, T. et al. Transient changes of the conformation of hemagglutinin of influenza virus at low pH detected by time-resolved circular dichroism spectroscopy. J. Biol. Chem. 272, 9764–9770 (1997).

    Article  CAS  Google Scholar 

  16. Chernomordik, L.V., 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  Google Scholar 

  17. Leikina, E. & Chernomordik, L.V. Reversible merger of membranes at the early stage of influenza hemagglutinin-mediated fusion. Mol. Biol. Cell 11, 2359–2371 (2000).

    Article  CAS  Google Scholar 

  18. Melikyan, G.B. et al. Evidence that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion. J. Cell Biol. 151, 413–423 (2000).

    Article  CAS  Google Scholar 

  19. Russell, C.J., Jardetzky, T.S. & Lamb, R.A. Membrane fusion machines of paramyxoviruses: capture of intermediates of fusion. EMBO J. 20, 4024–4034 (2001).

    Article  CAS  Google Scholar 

  20. Stegmann, T., White, J.M. & Helenius, A. Intermediates in influenza induced membrane fusion. EMBO J. 9, 4231–4241 (1990).

    Article  CAS  Google Scholar 

  21. Tamm, L.K. Hypothesis: spring-loaded boomerang mechanism of influenza hemagglutinin-mediated membrane fusion. Biochim. Biophys. Acta 1614, 14–23 (2003).

    Article  CAS  Google Scholar 

  22. Earp, L.J., Delos, S.E., Park, H.E. & White, J.M. The many mechanisms of viral membrane fusion proteins. Curr. Top. Microbiol. Immunol. 285, 25–66 (2005).

    CAS  PubMed  Google Scholar 

  23. Zaitseva, E., Mittal, A., Griffin, D.E. & Chernomordik, L.V. Class II fusion protein of alphaviruses drives membrane fusion through the same pathway as class I proteins. J. Cell Biol. 169, 167–177 (2005).

    Article  CAS  Google Scholar 

  24. Melikyan, G.B., Brener, S.A., Ok, D.C. & Cohen, F.S. Inner but not outer membrane leaflets control the transition from glycosylphosphatidylinositol-anchored influenza hemagglutinin-induced hemifusion to full fusion. J. Cell Biol. 136, 995–1005 (1997).

    Article  CAS  Google Scholar 

  25. Brydon, E.W., Smith, H. & Sweet, C. Influenza A virus-induced apoptosis in bronchiolar epithelial (NCI-H292) cells limits pro-inflammatory cytokine release. J. Gen. Virol. 84, 2389–2400 (2003).

    Article  CAS  Google Scholar 

  26. Blissard, G.W. & Wenz, J.R. Baculovirus gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion. J. Virol. 66, 6829–6835 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Chernomordik, L., Leikina, E. & Zimmerberg, J. Control of baculovirus gp64-induced syncytia formation by membrane lipid composition. J. Virol. 69, 3049–3058 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 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  Google Scholar 

  29. Gibbons, D.L. et al. Conformational change and protein-protein interactions of the fusion protein of Semliki Forest virus. Nature 427, 320–325 (2004).

    Article  CAS  Google Scholar 

  30. Wang, W., Cole, A.M., Hong, T., Waring, A.J. & Lehrer, R.I. Retrocyclin, an antiretroviral θ-defensin, is a lectin. J. Immunol. 170, 4708–4716 (2003).

    Article  CAS  Google Scholar 

  31. Schlessinger, J. et al. Lateral transport on cell membranes: mobility of concanavalin A receptors on myoblasts. Proc. Natl. Acad. Sci. USA 73, 2409–2413 (1976).

    Article  CAS  Google Scholar 

  32. Golan, D.E., Brown, C.S., Cianci, C.M., Furlong, S.T. & Caulfield, J.P. Schistosomula of Schistosoma mansoni use lysophosphatidylcholine to lyse adherent human red blood cells and immobilize red cell membrane components. J. Cell Biol. 103, 819–828 (1986).

    Article  CAS  Google Scholar 

  33. Crouch, E., Hartshorn, K. & Ofek, I. Collectins and pulmonary innate immunity. Immunol. Rev. 173, 52–65 (2000).

    Article  CAS  Google Scholar 

  34. Kase, T. et al. Human mannan-binding lectin inhibits the infection of influenza A virus without complement. Immunology 97, 385–392 (1999).

    Article  CAS  Google Scholar 

  35. Quinones-Mateu, M.E. et al. Human epithelial β-defensins 2 and 3 inhibit HIV-1 replication. AIDS 17, F39–F48 (2003).

    Article  CAS  Google Scholar 

  36. Schutte, B.C. & McCray, P.B., Jr. β-defensins in lung host defense. Annu. Rev. Physiol. 64, 709–748 (2002).

    Article  CAS  Google Scholar 

  37. Chernomordik, L.V. & Kozlov, M.M. Protein-lipid interplay in fusion and fission of biological membranes. Annu. Rev. Biochem. 72, 175–207 (2003).

    Article  CAS  Google Scholar 

  38. Leikina, E. et al. Influenza hemagglutinins outside of the contact zone are necessary for fusion pore expansion. J. Biol. Chem. 279, 26526–26532 (2004).

    Article  CAS  Google Scholar 

  39. Levroney, E.L. et al. Novel innate immune functions for galectin-1: galectin-1 inhibits cell fusion by nipah virus envelope glycoproteins and augments dendritic cell secretion of proinflammatory cytokines. J. Immunol. 175, 413–420 (2005).

    Article  CAS  Google Scholar 

  40. Thiel, S., Holmskov, U., Hviid, L., Laursen, S.B. & Jensenius, J.C. The concentration of the C-type lectin, mannan-binding protein, in human plasma increases during an acute phase response. Clin. Exp. Immunol. 90, 31–35 (1992).

    Article  CAS  Google Scholar 

  41. Kandel, R. & Hartshorn, K.L. Novel strategies for prevention and treatment of influenza. Expert Opin. Ther. Targets 9, 1–22 (2005).

    Article  CAS  Google Scholar 

  42. Tecle, T., White, M.R. & Hartshorn, K.L. Innate immunity to influenza A virus infection. Curr. Resp. Med. Rev. 1, 127–145 (2005).

    Article  CAS  Google Scholar 

  43. Eisen, D.P. & Minchinton, R.M. Impact of mannose-binding lectin on susceptibility to infectious diseases. Clin. Infect. Dis. 37, 1496–1505 (2003).

    Article  CAS  Google Scholar 

  44. Sawaki, K. et al. High concentration of β-defensin-2 in oral squamous cell carcinoma. Anticancer Res. 22, 2103–2107 (2002).

    CAS  PubMed  Google Scholar 

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

    Google Scholar 

  46. Mahy, B.W.J. & Kangro, H.O. Virology Methods Manual (Academic Press, San Diego, 1996).

    Google Scholar 

  47. Hoekstra, D., de Boer, 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. Doxsey, S.J., Sambrook, J., Helenius, A. & White, J. An efficient method for introducing macromolecules into living cells. J. Cell Biol. 101, 19–27 (1985).

    Article  CAS  Google Scholar 

  49. Hierholzer, J.C., Castells, E., Banks, G.G., Bryan, J.A. & McEwen, C.T. Sensitivity of NCI-H292 human lung mucoepidermoid cells for respiratory and other human viruses. J. Clin. Microbiol. 31, 1504–1510 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Koppel, D.E., Axelrod, D., Schlessinger, J., Elson, E.L. & Webb, W.W. Dynamics of fluorescence marker concentration as a probe of mobility. Biophys. J. 16, 1315–1329 (1976).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Kozlov, G. Melikyan, B. Podbilewicz, E. Zaitseva and J. Zimmerberg and H. Huang for discussions; and L. Bezrukova for advice on viral titer assay. Supported by the Intramural Research Program of the National Institutes of Health, National Institute of Child Health and Human Development, and by the National Institutes of Health (RR 20004 to J.A.L. and AI056921 to R.I.L.). The UCLA Functional Proteomics Center was established and equipped by a grant to the University of California at Los Angeles from the W.M. Keck Foundation.

Author information

Authors and Affiliations

  1. Section on Membrane Biology, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, Bethesda, 20892-1855, Maryland, USA

    Eugenia Leikina, Helene Delanoe-Ayari, Kamran Melikov, Andrew Chen & Leonid V Chernomordik

  2. Laboratory of Cell Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, 20892-1855, Maryland, USA

    Myoung-Soon Cho

  3. David Geffen School of Medicine at UCLA, University of California at Los Angeles, Los Angeles, 90095-1690, California, USA

    Alan J Waring, Wei Wang, Joseph A Loo & Robert I Lehrer

  4. Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, 90095-1690, California, USA

    Yongming Xie & Joseph A Loo

Authors
  1. Eugenia Leikina
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Helene Delanoe-Ayari
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kamran Melikov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Myoung-Soon Cho
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andrew Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alan J Waring
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yongming Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Joseph A Loo
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Robert I Lehrer
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Leonid V Chernomordik
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Leonid V Chernomordik.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

RC2 in concentrations up to 200 μg/ml is not toxic for MDCK and HAb2 cells. (PDF 34 kb)

Supplementary Fig. 2

RC2 does not affect membrane contact. (PDF 389 kb)

Supplementary Fig. 3

RC2 inhibits lipid mixing between influenza virus and RBC. (PDF 123 kb)

Supplementary Fig. 4

RC2 inhibits restricted hemifusion. (PDF 38 kb)

Supplementary Fig. 5

RC2 does not alter the pH-dependence of fusion. (PDF 47 kb)

Supplementary Fig. 6

Fusion inhibition by RC2 involves its reversible interactions with cell surface carbohydrates. (PDF 20 kb)

Supplementary Fig. 7

Multivalency of RC2. (PDF 226 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Leikina, E., Delanoe-Ayari, H., Melikov, K. et al. Carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins. Nat Immunol 6, 995–1001 (2005). https://doi.org/10.1038/ni1248

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni1248

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