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Structure of the dengue virus envelope protein after membrane fusion

Abstract

Dengue virus enters a host cell when the viral envelope glycoprotein, E, binds to a receptor and responds by conformational rearrangement to the reduced pH of an endosome. The conformational change induces fusion of viral and host-cell membranes. A three-dimensional structure of the soluble E ectodomain (sE) in its trimeric, postfusion state reveals striking differences from the dimeric, prefusion form. The elongated trimer bears three ‘fusion loops’ at one end, to insert into the host-cell membrane. Their structure allows us to model directly how these fusion loops interact with a lipid bilayer. The protein folds back on itself, directing its carboxy terminus towards the fusion loops. We propose a fusion mechanism driven by essentially irreversible conformational changes in E and facilitated by fusion-loop insertion into the outer bilayer leaflet. Specific features of the folded-back structure suggest strategies for inhibiting flavivirus entry.

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References

  1. 1

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

  2. 2

    Wilson, I. A., Skehel, J. J. & Wiley, D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 Å resolution. Nature 289, 366–373 (1981)

  3. 3

    Baker, K. A., Dutch, R. E., Lamb, R. A. & Jardetzky, T. S. Structural basis for paramyxovirus-mediated membrane fusion. Mol. Cell 3, 309–319 (1999)

  4. 4

    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)

  5. 5

    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)

  6. 6

    Bullough, P. A., Hughson, F. M., Skehel, J. J. & Wiley, D. C. Structure of influenza haemagglutinin at the pH of membrane fusion. Nature 371, 37–43 (1994)

  7. 7

    Chen, J., Skehel, J. J. & Wiley, D. C. N- and C-terminal residues combine in the fusion-pH influenza hemagglutinin HA(2) subunit to form an N cap that terminates the triple-stranded coiled coil. Proc. Natl Acad. Sci. USA 96, 8967–8972 (1999)

  8. 8

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

  9. 9

    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)

  10. 10

    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, 6986–6991 (2003)

  11. 11

    Allison, S. L. et al. Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH. J. Virol. 69, 695–700 (1995)

  12. 12

    Ferlenghi, I. et al. Molecular organization of a recombinant subviral particle from tick-borne encephalitis virus. Mol. Cell 7, 593–602 (2001)

  13. 13

    Kuhn, R. J. et al. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 108, 717–725 (2002)

  14. 14

    Allison, S. L., Schalich, J., Stiasny, K., Mandl, C. W. & Heinz, F. X. Mutational evidence for an internal fusion peptide in flavivirus envelope protein E. J. Virol. 75, 4268–4275 (2001)

  15. 15

    Levy-Mintz, P. & Kielian, M. Mutagenesis of the putative fusion domain of the Semliki Forest virus spike protein. J. Virol. 65, 4292–4300 (1991)

  16. 16

    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)

  17. 17

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

  18. 18

    Stiasny, K., Allison, S. L., Schalich, J. & Heinz, F. X. Membrane interactions of the tick-borne encephalitis virus fusion protein E at low pH. J. Virol. 76, 3784–3790 (2002)

  19. 19

    Wimley, W. C. & White, S. H. Partitioning of tryptophan side-chain analogs between water and cyclohexane. Biochemistry 31, 12813–12818 (1992)

  20. 20

    Allison, S. L., Stiasny, K., Stadler, K., Mandl, C. W. & Heinz, F. X. Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E. J. Virol. 73, 5605–5612 (1999)

  21. 21

    Zhang, W. et al. Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nature Struct. Biol. 10, 907–912 (2003)

  22. 22

    Crill, W. D. & Roehrig, J. T. Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J. Virol. 75, 7769–7773 (2001)

  23. 23

    Jennings, A. D. et al. Analysis of a yellow fever virus isolated from a fatal case of vaccine-associated human encephalitis. J. Infect. Dis. 169, 512–518 (1994)

  24. 24

    Lobigs, M. et al. Host cell selection of Murray Valley encephalitis virus variants altered at an RGD sequence in the envelope protein and in mouse virulence. Virology 176, 587–595 (1990)

  25. 25

    Holzmann, H., Heinz, F. X., Mandl, C. W., Guirakhoo, F. & Kunz, C. A single amino acid substitution in envelope protein E of tick-borne encephalitis virus leads to attenuation in the mouse model. J. Virol. 64, 5156–5159 (1990)

  26. 26

    Jiang, W. R., Lowe, A., Higgs, S., Reid, H. & Gould, E. A. Single amino acid codon changes detected in louping ill virus antibody-resistant mutants with reduced neurovirulence. J. Gen. Virol. 74, 931–935 (1993)

  27. 27

    Gao, G. F., Hussain, M. H., Reid, H. W. & Gould, E. A. Identification of naturally occurring monoclonal antibody escape variants of louping ill virus. J. Gen. Virol. 75, 609–614 (1994)

  28. 28

    Cecilia, D. & Gould, E. A. Nucleotide changes responsible for loss of neuroinvasiveness in Japanese encephalitis virus neutralization-resistant mutants. Virology 181, 70–77 (1991)

  29. 29

    Chen, Y. et al. Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate. Nature Med. 3, 866–871 (1997)

  30. 30

    Navarro-Sanchez, E. et al. Dendritic-cell-specific ICAM3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses. EMBO Rep. 4, 1–6 (2003)

  31. 31

    Tassaneetrithep, B. et al. DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J. Exp. Med. 197, 823–829 (2003)

  32. 32

    Stiasny, K., Allison, S. L., Marchler-Bauer, A., Kunz, C. & Heinz, F. X. Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus. J. Virol. 70, 8142–8147 (1996)

  33. 33

    Chan, D. C. & Kim, P. S. HIV entry and its inhibition. Cell 93, 681–684 (1998)

  34. 34

    Kuzmin, P. I., Zimmerberg, J., Chizmadzhev, Y. A. & Cohen, F. S. A quantitative model for membrane fusion based on low-energy intermediates. Proc. Natl Acad. Sci. USA 98, 7235–7240 (2001)

  35. 35

    Razinkov, V. I., Melikyan, G. B. & Cohen, F. S. Hemifusion between cells expressing hemagglutinin of influenza virus and planar membranes can precede the formation of fusion pores that subsequently fully enlarge. Biophys. J. 77, 3144–3151 (1999)

  36. 36

    Kozlov, M. M. & Chernomordik, L. V. A mechanism of protein-mediated fusion: coupling between refolding of the influenza hemagglutinin and lipid rearrangements. Biophys. J. 75, 1384–1396 (1998)

  37. 37

    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)

  38. 38

    Han, X., Bushweller, J. H., Cafiso, D. S. & Tamm, L. K. Membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin. Nature Struct. Biol. 8, 715–720 (2001)

  39. 39

    Ito, H., Watanabe, S., Sanchez, A., Whitt, M. A. & Kawaoka, Y. Mutational analysis of the putative fusion domain of Ebola virus glycoprotein. J. Virol. 73, 8907–8912 (1999)

  40. 40

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

  41. 41

    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)

  42. 42

    Dutch, R. E. & Lamb, R. A. Deletion of the cytoplasmic tail of the fusion protein of the paramyxovirus simian virus 5 affects fusion pore enlargement. J. Virol. 75, 5363–5369 (2001)

  43. 43

    Baldwin, C. E., Sanders, R. W. & Berkhout, B. Inhibiting HIV-1 entry with fusion inhibitors. Curr. Med. Chem. 10, 1633–1642 (2003)

  44. 44

    Kilby, J. M. et al. Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry. Nature Med. 4, 1302–1307 (1998)

  45. 45

    Hahn, Y. S. et al. Nucleotide sequence of dengue 2 RNA and comparison of the encoded proteins with those of other flaviviruses. Virology 162, 167–180 (1988)

  46. 46

    Ivy, J., Nakano, E. & Clements, D. Subunit immunogenic composition against dengue infection. US Patent 6,165,477 (1997)

  47. 47

    Cuzzubbo, A. J. et al. Use of recombinant envelope proteins for serological diagnosis of dengue virus infection in an immunochromatographic assay. Clin. Diagn. Lab. Immunol. 8, 1150–1155 (2001)

  48. 48

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  49. 49

    Navaza, J. Implementation of molecular replacement in AMoRe. Acta Crystallogr. D 57, 1367–1372 (2001)

  50. 50

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

  51. 51

    Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

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Acknowledgements

We thank staff at BioCARS beamline 14-BM-C at the Advanced Photon Source (Argonne National Laboratory). We thank J. Zimmerberg, F. Rey and F. Heinz for discussions, and T. Walz and Y. Cheng for guidance on EM experiments. This work was supported by a long-term fellowship to Y.M. from the Human Frontier Science Program Organization, and by an NIH. grant to S.C.H., who is a Howard Hughes Medical Institute Investigator.

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Correspondence to Stephen C. Harrison.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Table : Crystallographic data and refinement statistics. (DOC 27 kb)

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Further reading

Figure 1: Structure of the dimer of dengue E soluble fragment (sE) in the mature virus particle.
Figure 2: Trimer formation and membrane insertion of dengue E protein.
Figure 3: Domain rearrangements in the dengue sE monomer during the transition to trimer.
Figure 4: The dengue sE trimer.
Figure 5: Proposed mechanism for fusion mediated by class II viral fusion proteins.

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