Flaviviruses infect their host cells by a membrane fusion reaction. In this study, we performed a functional analysis of the membrane fusion properties of West Nile virus (WNV) with liposomal target membranes. Membrane fusion was monitored continuously using a lipid mixing assay involving the fluorophore, pyrene. Fusion of WNV with liposomes occurred on the timescale of seconds and was strictly dependent on mildly acidic pH. Optimal fusion kinetics were observed at pH 6.3, the threshold for fusion being pH 6.9. Preincubation of the virus alone at pH 6.3 resulted in a rapid loss of fusion capacity. WNV fusion activity is strongly promoted by the presence of cholesterol in the target membrane. Furthermore, we provide direct evidence that cleavage of prM to M is a requirement for fusion activity of WNV.


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  1. 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. [Google Scholar]
  2. Corver, J., Ortiz, A., Allison, S. L., Schalich, J., Heinz, F. X. & Wilschut, J.(2000). Membrane fusion activity of tick-borne encephalitis virus and recombinant subviral particles in a liposomal model system. Virology 269, 37–46.[CrossRef] [Google Scholar]
  3. Elshuber, S., Allison, S. L., Heinz, F. X. & Mandl, C. W.(2003). Cleavage of protein prM is necessary for infection of BHK-21 cells by tick-borne encephalitis virus. J Gen Virol 84, 183–191.[CrossRef] [Google Scholar]
  4. Gollins, S. W. & Porterfield, J. S.(1986). pH-dependent fusion between the flavivirus West Nile and liposomal model membranes. J Gen Virol 67, 157–166.[CrossRef] [Google Scholar]
  5. Guirakhoo, F., Heinz, F. X., Mandl, C. W., Holzmann, H. & Kunz, C.(1991). Fusion activity of flaviviruses: comparison of mature and immature (prM-containing) tick-borne encephalitis virions. J Gen Virol 72, 1323–1329.[CrossRef] [Google Scholar]
  6. Kielian, M. & Rey, F. A.(2006). Virus membrane-fusion proteins: more than one way to make a hairpin. Nat Rev Microbiol 4, 67–76.[CrossRef] [Google Scholar]
  7. Kuhn, R. J., Zhang, W., Rossmann, M. G., Pletnev, S. V., Corver, J., Lenches, E., Jones, C. T., Mukhopadhyay, S., Chipman, P. R. & other authors(2002). Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 108, 717–725.[CrossRef] [Google Scholar]
  8. Lindenbach, D. & Rice, C. M.(2001).Flaviviridae: the viruses and their replication. In Fields Virology, 4th edn, pp. 991–1041. Edited by D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, B. Roizman & S. E. Strauss. Philadelphia, PA: Lippincott Williams and Wilkins.
  9. Medigeshi, G. R., Hirsch, A. J., Streblow, D. N., Nikolich-Zugich, J. & Nelson, J. A.(2008). West Nile virus entry requires cholesterol-rich membrane microdomains and is independent of αvβ3 integrin. J Virol 82, 5212–5219.[CrossRef] [Google Scholar]
  10. 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. [Google Scholar]
  11. 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] [Google Scholar]
  12. 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. [Google Scholar]
  13. Stadler, K., Allison, S. L., Schalich, J. & Heinz, F. X.(1997). Proteolytic activation of tick-borne encephalitis virus by furin. J Virol 71, 8475–8481. [Google Scholar]
  14. Stiasny, K., Koessl, C. & Heinz, F. X.(2003). Involvement of lipids in different steps of the flavivirus fusion mechanism. J Virol 77, 7856–7862.[CrossRef] [Google Scholar]
  15. Takahashi, S., Kasai, K., Hatsuzawa, K., Kitamura, N., Misumi, Y., Ikehara, Y., Murakami, K. & Nakayama, K.(1993). A mutation of furin causes the lack of precursor-processing activity in human colon carcinoma LoVo cells. Biochem Biophys Res Commun 195, 1019–1026.[CrossRef] [Google Scholar]
  16. Umashankar, M., Sanchez-San, M. 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] [Google Scholar]
  17. Wengler, G. & Wengler, G.(1989). Cell-associated West Nile flavivirus is covered with E+pre-M protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release. J Virol 63, 2521–2526. [Google Scholar]
  18. Yu, I. M., Zhang, W., Holdaway, H. A., Li, L., Kostyuchenko, V. A., Chipman, P. R., Kuhn, R. J., Rossmann, M. G. & Chen, J.(2008). Structure of the immature dengue virus at low pH primes proteolytic maturation. Science 319, 1834–1837.[CrossRef] [Google Scholar]
  19. Zybert, I. A., van der Ende-Metselaar, H., Wilschut, J. & Smit, J. M.(2008). Functional importance of dengue virus maturation: infectious properties of immature virions. J Gen Virol 89, 3047–3051.[CrossRef] [Google Scholar]

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