1887

Abstract

Chikungunya virus (CHIKV) is a rapidly spreading, enveloped alphavirus causing fever, rash and debilitating polyarthritis. No specific treatment or vaccines are available to treat or prevent infection. For the rational design of vaccines and antiviral drugs, it is imperative to understand the molecular mechanisms involved in CHIKV infection. A critical step in the life cycle of CHIKV is fusion of the viral membrane with a host cell membrane. Here, we elucidate this process using ensemble-averaging liposome–virus fusion studies, in which the fusion behaviour of a large virus population is measured, and a newly developed microscopy-based single-particle assay, in which the fusion kinetics of an individual particle can be visualised. The combination of these approaches allowed us to obtain detailed insight into the kinetics, lipid dependency and pH dependency of hemifusion. We found that CHIKV fusion is strictly dependent on low pH, with a threshold of pH 6.2 and optimal fusion efficiency below pH 5.6. At this pH, CHIKV fuses rapidly with target membranes, with typically half of the fusion occurring within 2 s after acidification. Cholesterol and sphingomyelin in the target membrane were found to strongly enhance the fusion process. By analysing our single-particle data using kinetic models, we were able to deduce that the number of rate-limiting steps occurring before hemifusion equals about three. To explain these data, we propose a mechanistic model in which multiple E1 fusion trimers are involved in initiating the fusion process.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.000144
2015-08-01
2019-12-06
Loading full text...

Full text loading...

/deliver/fulltext/jgv/96/8/2122.html?itemId=/content/journal/jgv/10.1099/vir.0.000144&mimeType=html&fmt=ahah

References

  1. Ahn A. , Gibbons D.L. , Kielian M. . ( 2002;). The fusion peptide of Semliki Forest virus associates with sterol-rich membrane domains. J Virol 76: 3267–3275 [CrossRef] [PubMed].
    [Google Scholar]
  2. Bernard E. , Solignat M. , Gay B. , Chazal N. , Higgs S. , Devaux C. , Briant L. . ( 2010;). Endocytosis of chikungunya virus into mammalian cells: role of clathrin and early endosomal compartments. PLoS One 5: e11479 [CrossRef] [PubMed].
    [Google Scholar]
  3. Böttcher C.S.F. , Van gent C.M. , Pries C. . ( 1961;). A rapid and sensitive submicro phosphorus determination. Annal Chim Acta 24: 203–204 [CrossRef].
    [Google Scholar]
  4. Brandenburg B. , Koudstaal W. , Goudsmit J. , Klaren V. , Tang C. , Bujny M.V. , Korse H.J. , Kwaks T. , Otterstrom J.J. , other authors . ( 2013;). Mechanisms of hemagglutinin targeted influenza virus neutralization. PLoS One 8: e80034 [CrossRef] [PubMed].
    [Google Scholar]
  5. Brandenberg O.F. , Magnus C. , Rusert P. , Regoes R.R. , Trkola A. . ( 2015;). Different infectivity of HIV-1 strains is linked to number of envelope trimers required for entry. PLoS Pathog 11: e1004595 [CrossRef] [PubMed].
    [Google Scholar]
  6. Bron R. , Wahlberg J.M. , Garoff H. , Wilschut J. . ( 1993;). Membrane fusion of Semliki Forest virus in a model system: correlation between fusion kinetics and structural changes in the envelope glycoprotein. EMBO J 12: 693–701 [PubMed].
    [Google Scholar]
  7. Burt F.J. , Rolph M.S. , Rulli N.E. , Mahalingam S. , Heise M.T. . ( 2012;). Chikungunya: a re-emerging virus. Lancet 379: 662–671 [CrossRef] [PubMed].
    [Google Scholar]
  8. Centers for Disease Control and Prevention ( 2015;). Chikungunya in the Americas http://www.cdc.gov/chikungunya/geo/americas.html. [Accessed 3 February 2015.].
    [Google Scholar]
  9. Chatterjee P.K. , Eng C.H. , Kielian M. . ( 2002;). Novel mutations that control the sphingolipid and cholesterol dependence of the Semliki Forest virus fusion protein. J Virol 76: 12712–12722 [CrossRef] [PubMed].
    [Google Scholar]
  10. Costello D.A. , Lee D.W. , Drewes J. , Vasquez K.A. , Kisler K. , Wiesner U. , Pollack L. , Whittaker G.R. , Daniel S. . ( 2012;). Influenza virus-membrane fusion triggered by proton uncaging for single particle studies of fusion kinetics. Anal Chem 84: 8480–8489 [CrossRef] [PubMed].
    [Google Scholar]
  11. Danieli T. , Pelletier S.L. , Henis Y.I. , White J.M. . ( 1996;). Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers. J Cell Biol 133: 559–569 [CrossRef] [PubMed].
    [Google Scholar]
  12. Enserink M. . ( 2007;). Infectious diseases. Chikungunya: no longer a third world disease. Science 318: 1860–1861 [CrossRef] [PubMed].
    [Google Scholar]
  13. Enserink M. . ( 2014;). Infectious diseases. Crippling virus set to conquer Western Hemisphere. Science 344: 678–679 [CrossRef] [PubMed].
    [Google Scholar]
  14. Fischer M. , Staples J.E. , Arboviral Diseases Branch, National Center for Emerging and Zoonotic Infectious Diseases, CDC . ( 2014;). Notes from the field: chikungunya virus spreads in the Americas - Caribbean and South America, 2013-2014. MMWR Morb Mortal Wkly Rep 63: 500–501 [PubMed].
    [Google Scholar]
  15. Floyd D.L. , Ragains J.R. , Skehel J.J. , Harrison S.C. , van Oijen A.M. . ( 2008;). Single-particle kinetics of influenza virus membrane fusion. Proc Natl Acad Sci U S A 105: 15382–15387 [CrossRef] [PubMed].
    [Google Scholar]
  16. Floyd D.L. , Harrison S.C. , van Oijen A.M. . ( 2010;). Analysis of kinetic intermediates in single-particle dwell-time distributions. Biophys J 99: 360–366 [CrossRef] [PubMed].
    [Google Scholar]
  17. Gay B. , Bernard E. , Solignat M. , Chazal N. , Devaux C. , Briant L. . ( 2012;). pH-dependent entry of chikungunya virus into Aedes albopictus cells. Infect Genet Evol 12: 1275–1281 [CrossRef] [PubMed].
    [Google Scholar]
  18. Gibbons D.L. , Ahn A. , Liao M. , Hammar L. , Cheng R.H. , Kielian M. . ( 2004a;). Multistep regulation of membrane insertion of the fusion peptide of Semliki Forest virus. J Virol 78: 3312–3318 [CrossRef] [PubMed].
    [Google Scholar]
  19. Gibbons D.L. , Vaney M.C. , Roussel A. , Vigouroux A. , Reilly B. , Lepault J. , Kielian M. , Rey F.A. . ( 2004b;). Conformational change and protein-protein interactions of the fusion protein of Semliki Forest virus. Nature 427: 320–325 [CrossRef] [PubMed].
    [Google Scholar]
  20. Glomb-Reinmund S. , Kielian M. . ( 1998;). The role of low pH and disulfide shuffling in the entry and fusion of Semliki Forest virus and Sindbis virus. Virology 248: 372–381 [CrossRef] [PubMed].
    [Google Scholar]
  21. Harrison S.C. . ( 2008;). Viral membrane fusion. Nat Struct Mol Biol 15: 690–698 [CrossRef] [PubMed].
    [Google Scholar]
  22. Hinterdorfer P. , Baber G. , Tamm L.K. . ( 1994;). Reconstitution of membrane fusion sites. A total internal reflection fluorescence microscopy study of influenza hemagglutinin-mediated membrane fusion. J Biol Chem 269: 20360–20368 [PubMed].
    [Google Scholar]
  23. Hoekstra D. , de Boer T. , Klappe K. , Wilschut J. . ( 1984;). Fluorescence method for measuring the kinetics of fusion between biological membranes. Biochemistry 23: 5675–5681 [CrossRef] [PubMed].
    [Google Scholar]
  24. Imai M. , Mizuno T. , Kawasaki K. . ( 2006;). Membrane fusion by single influenza hemagglutinin trimers. Kinetic evidence from image analysis of hemagglutinin-reconstituted vesicles. J Biol Chem 281: 12729–12735 [CrossRef] [PubMed].
    [Google Scholar]
  25. Ivanovic T. , Choi J.L. , Whelan S.P. , van Oijen A.M. , Harrison S.C. . ( 2013;). Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates. eLife 2: e00333 [CrossRef] [PubMed].
    [Google Scholar]
  26. Kielian M. . ( 2014;). Mechanisms of virus membrane fusion proteins. Annu Rev Virol 1: 171–189 [CrossRef].
    [Google Scholar]
  27. Kielian M.C. , Helenius A. . ( 1984;). Role of cholesterol in fusion of Semliki Forest virus with membranes. J Virol 52: 281–283 [PubMed].
    [Google Scholar]
  28. Kielian M. , Chanel-Vos C. , Liao M. . ( 2010;). Alphavirus entry and membrane fusion. Viruses 2: 796–825 [CrossRef] [PubMed].
    [Google Scholar]
  29. Klimjack M.R. , Jeffrey S. , Kielian M. . ( 1994;). Membrane and protein interactions of a soluble form of the Semliki Forest virus fusion protein. J Virol 68: 6940–6946 [PubMed].
    [Google Scholar]
  30. Kolter T. , Sandhoff K. . ( 2010;). Lysosomal degradation of membrane lipids. FEBS Lett 584: 1700–1712 [CrossRef] [PubMed].
    [Google Scholar]
  31. Kucharz E.J. , Cebula-Byrska I. . ( 2012;). Chikungunya fever. Eur J Intern Med 23: 325–329 [CrossRef] [PubMed].
    [Google Scholar]
  32. Laine R. , Söderlund H. , Renkonen O. . ( 1973;). Chemical composition of Semliki forest virus. Intervirology 1: 110–118 [CrossRef] [PubMed].
    [Google Scholar]
  33. Lescar J. , Roussel A. , Wien M.W. , Navaza J. , Fuller S.D. , Wengler G. , Wengler G. , Rey F.A. . ( 2001;). The fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal pH. Cell 105: 137–148 [CrossRef] [PubMed].
    [Google Scholar]
  34. Leung J.Y. , Ng M.M. , Chu J.J. . ( 2011;). Replication of alphaviruses: a review on the entry process of alphaviruses into cells. Adv Virol 2011: 249640 [CrossRef] [PubMed].
    [Google Scholar]
  35. Li L. , Jose J. , Xiang Y. , Kuhn R.J. , Rossmann M.G. . ( 2010;). Structural changes of envelope proteins during alphavirus fusion. Nature 468: 705–708 [CrossRef] [PubMed].
    [Google Scholar]
  36. Lu Y.E. , Cassese T. , Kielian M. . ( 1999;). The cholesterol requirement for Sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence. J Virol 73: 4272–4278 [PubMed].
    [Google Scholar]
  37. Melikyan G.B. , Barnard R.J. , Abrahamyan L.G. , Mothes W. , Young J.A. . ( 2005;). Imaging individual retroviral fusion events: from hemifusion to pore formation and growth. Proc Natl Acad Sci U S A 102: 8728–8733 [CrossRef] [PubMed].
    [Google Scholar]
  38. Moesby L. , Corver J. , Erukulla R.K. , Bittman R. , Wilschut J. . ( 1995;). Sphingolipids activate membrane fusion of Semliki Forest virus in a stereospecific manner. Biochemistry 34: 10319–10324 [CrossRef] [PubMed].
    [Google Scholar]
  39. Mooney J.J. , Dalrymple J.M. , Alving C.R. , Russell P.K. . ( 1975;). Interaction of Sindbis virus with liposomal model membranes. J Virol 15: 225–231 [PubMed].
    [Google Scholar]
  40. Nieva J.L. , Bron R. , Corver J. , Wilschut J. . ( 1994;). Membrane fusion of Semliki Forest virus requires sphingolipids in the target membrane. EMBO J 13: 2797–2804 [PubMed].
    [Google Scholar]
  41. Niles W.D. , Cohen F.S. . ( 1991;). Fusion of influenza virions with a planar lipid membrane detected by video fluorescence microscopy. J Gen Physiol 97: 1101–1119 [CrossRef] [PubMed].
    [Google Scholar]
  42. Nollert P. , Kiefer H. , Jähnig F. . ( 1995;). Lipid vesicle adsorption versus formation of planar bilayers on solid surfaces. Biophys J 69: 1447–1455 [CrossRef] [PubMed].
    [Google Scholar]
  43. Pal R. , Barenholz Y. , Wagner R.R. . ( 1988;). Pyrene phospholipid as a biological fluorescent probe for studying fusion of virus membrane with liposomes. Biochemistry 27: 30–36 [CrossRef] [PubMed].
    [Google Scholar]
  44. Powers A.M. , Brault A.C. , Shirako Y. , Strauss E.G. , Kang W. , Strauss J.H. , Weaver S.C. . ( 2001;). Evolutionary relationships and systematics of the alphaviruses. J Virol 75: 10118–10131 [CrossRef] [PubMed].
    [Google Scholar]
  45. Ross R.W. . ( 1956;). A laboratory technique for studying the insect transmission of animal viruses, employing a bat-wing membrane, demonstrated with two African viruses. J Hyg (Lond) 54: 192–200 [CrossRef] [PubMed].
    [Google Scholar]
  46. Samsonov A.V. , Chatterjee P.K. , Razinkov V.I. , Eng C.H. , Kielian M. , Cohen F.S. . ( 2002;). Effects of membrane potential and sphingolipid structures on fusion of Semliki Forest virus. J Virol 76: 12691–12702 [CrossRef] [PubMed].
    [Google Scholar]
  47. Sánchez-San Martín C. , Sosa H. , Kielian M. . ( 2008;). A stable prefusion intermediate of the alphavirus fusion protein reveals critical features of class II membrane fusion. Cell Host Microbe 4: 600–608 [CrossRef] [PubMed].
    [Google Scholar]
  48. Sánchez-San Martín C. , Nanda S. , Zheng Y. , Fields W. , Kielian M. . ( 2013;). Cross-inhibition of chikungunya virus fusion and infection by alphavirus E1 domain III proteins. J Virol 87: 7680–7687 [CrossRef] [PubMed].
    [Google Scholar]
  49. Schwartz O. , Albert M.L. . ( 2010;). Biology and pathogenesis of chikungunya virus. Nat Rev Microbiol 8: 491–500 [CrossRef] [PubMed].
    [Google Scholar]
  50. Smit J.M. , Bittman R. , Wilschut J. . ( 1999;). Low-pH-dependent fusion of Sindbis virus with receptor-free cholesterol- and sphingolipid-containing liposomes. J Virol 73: 8476–8484 [PubMed].
    [Google Scholar]
  51. Sourisseau M. , Schilte C. , Casartelli N. , Trouillet C. , Guivel-Benhassine F. , Rudnicka D. , Sol-Foulon N. , Le Roux K. , Prevost M.C. , other authors . ( 2007;). Characterization of reemerging chikungunya virus. PLoS Pathog 3: e89 [CrossRef] [PubMed].
    [Google Scholar]
  52. Strauss J.H. , Strauss E.G. . ( 1994;). The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 58: 491–562 [PubMed].
    [Google Scholar]
  53. Thiberville S.D. , Moyen N. , Dupuis-Maguiraga L. , Nougairede A. , Gould E.A. , Roques P. , de Lamballerie X. . ( 2013;). Chikungunya fever: epidemiology, clinical syndrome, pathogenesis and therapy. Antiviral Res 99: 345–370 [CrossRef] [PubMed].
    [Google Scholar]
  54. Thompson B.S. , Moesker B. , Smit J.M. , Wilschut J. , Diamond M.S. , Fremont D.H. . ( 2009;). A therapeutic antibody against West Nile virus neutralizes infection by blocking fusion within endosomes. PLoS Pathog 5: e1000453 [CrossRef] [PubMed].
    [Google Scholar]
  55. Tomasello D. , Schlagenhauf P. . ( 2013;). Chikungunya and dengue autochthonous cases in Europe, 2007-2012. Travel Med Infect Dis 11: 274–284 [CrossRef] [PubMed].
    [Google Scholar]
  56. Tsetsarkin K.A. , Vanlandingham D.L. , McGee C.E. , Higgs S. . ( 2007;). A single mutation in chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog 3: e201 [CrossRef] [PubMed].
    [Google Scholar]
  57. Tsetsarkin K.A. , McGee C.E. , Higgs S. . ( 2011;). Chikungunya virus adaptation to aedes Aedes albopictus does not correlate with acquisition of cholesterol dependence or decreased pH threshold for fusion reaction. Virol J 8: 376.[CrossRef]
    [Google Scholar]
  58. Umashankar M. , Sánchez-San Martín C. , Liao M. , Reilly B. , Guo A. , Taylor G. , Kielian M. . ( 2008;). Differential cholesterol binding by class II fusion proteins determines membrane fusion properties. J Virol 82: 9245–9253 [CrossRef] [PubMed].
    [Google Scholar]
  59. van der Schaar H.M. , Rust M.J. , Waarts B.L. , van der Ende-Metselaar H. , Kuhn R.J. , Wilschut J. , Zhuang X. , Smit J.M. . ( 2007;). Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking. J Virol 81: 12019–12028 [CrossRef] [PubMed].
    [Google Scholar]
  60. van Meer G. , Voelker D.R. , Feigenson G.W. . ( 2008;). Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol 9: 112–124 [CrossRef] [PubMed].
    [Google Scholar]
  61. Vancini R. , Wang G. , Ferreira D. , Hernandez R. , Brown D.T. . ( 2013;). Alphavirus genome delivery occurs directly at the plasma membrane in a time- and temperature-dependent process. J Virol 87: 4352–4359 [CrossRef] [PubMed].
    [Google Scholar]
  62. Voss J.E. , Vaney M.C. , Duquerroy S. , Vonrhein C. , Girard-Blanc C. , Crublet E. , Thompson A. , Bricogne G. , Rey F.A. . ( 2010;). Glycoprotein organization of Chikungunya virus particles revealed by X-ray crystallography. Nature 468: 709–712 [CrossRef] [PubMed].
    [Google Scholar]
  63. Waarts B.L. , Bittman R. , Wilschut J. . ( 2002;). Sphingolipid and cholesterol dependence of alphavirus membrane fusion. Lack of correlation with lipid raft formation in target liposomes. J Biol Chem 277: 38141–38147 [CrossRef] [PubMed].
    [Google Scholar]
  64. Waarts B.L. , Smit J.M. , Aneke O.J. , McInerney G.M. , Liljeström P. , Bittman R. , Wilschut J. . ( 2005;). Reversible acid-induced inactivation of the membrane fusion protein of Semliki Forest virus. J Virol 79: 7942–7948 [CrossRef] [PubMed].
    [Google Scholar]
  65. Wahlberg J.M. , Garoff H. . ( 1992;). 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 [CrossRef] [PubMed].
    [Google Scholar]
  66. Wahlberg J.M. , Boere W.A. , Garoff H. . ( 1989;). The heterodimeric association between the membrane proteins of Semliki Forest virus changes its sensitivity to low pH during virus maturation. J Virol 63: 4991–4997 [PubMed].
    [Google Scholar]
  67. Wahlberg J.M. , Bron R. , Wilschut J. , Garoff H. . ( 1992;). Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein. J Virol 66: 7309–7318 [PubMed].
    [Google Scholar]
  68. Wengler G. , Koschinski A. , Wengler G. , Repp H. . ( 2004;). During entry of alphaviruses, the E1 glycoprotein molecules probably form two separate populations that generate either a fusion pore or ion-permeable pores. J Gen Virol 85: 1695–1701 [CrossRef] [PubMed].
    [Google Scholar]
  69. Wessels L. , Elting M.W. , Scimeca D. , Weninger K. . ( 2007;). Rapid membrane fusion of individual virus particles with supported lipid bilayers. Biophys J 93: 526–538 [CrossRef] [PubMed].
    [Google Scholar]
  70. White J. , Helenius A. . ( 1980;). pH-dependent fusion between the Semliki Forest virus membrane and liposomes. Proc Natl Acad Sci U S A 77: 3273–3277 [CrossRef] [PubMed].
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.000144
Loading
/content/journal/jgv/10.1099/vir.0.000144
Loading

Data & Media loading...

Supplements

Supplementary Data



PDF

Supplementary Data



Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error