病毒感染細胞的第一步是附著在細胞表面的分子，這一個步驟決定了許多病毒感染細胞組織的趨性和病毒的致病力。病毒感染宿主細胞時會與細胞表面上的蛋白質接受器，多醣體或酯質層鍵結後再進入細胞。在先前的研究報告中，日本腦炎是利用外套膜蛋白與多醣體結合的機制進入細胞。因此，細胞表面上的肝素與日本腦炎病毒外套膜蛋白間的作用關係對於病毒進入細胞機制中扮演著重要的角色。

位於外套膜蛋白中的第三區塊N端前的279-297位置中含有四個帶正電的殘基。而細胞表面的肝素為帶有大量負電荷的化學分子，因此我們逐一將這四個帶正電的殘基定點突變，希望能找到主要的肝素鍵結的位置。並利用核磁共振和螢光的技術去檢測日本腦炎第三區塊以及E261-402蛋白和肝素之間的鍵結。這些結構的資料將提供非常重要的資訊作為循理性的疫苗設計與建立由肝素所衍生的抑制藥物的設計平台。

The initial binding of a virus to a target cell is a critical determinant of cell and tissue tropism, and thus of pathogenesis. Flaviviruses have a relatively simple structure with only a single envelope protein, making it likely that the motif responsible for target cell binding is expressed by the ectodomain of the envelope protein. Heparan sulfate (HS) are present in the extracellular matrix and/or tissue cell surface and involved in infection of JEV virus. Thus, the heparin binding activity of JEV may play an important role in its function on cellular surfaces.

In the present study, we attempted to identify which of the basic residue contribute to the heparin binding of E279 to E297. We had created several mutants by substituting basic residues (K279, K286, R288, K290) with alanine in the putative heparin-binding regions. We also analyze the heparin binding affinity through fluorescence and NMR methods. The completion of this study will provide a structural basis for design of vaccines.

1. Bruke, D. S., and Monath, T. P. (2001) Field Virology 4th Ed, 1043-1125
2. Rey, F. A., Heinz, F. X., Mandl, C., Kunz, C., and Harrison, S. C. (1995) Nature 375(6529), 291-298
3. Kuhn, R. J., Zhang, W., Rossmann, M. G., Pletnev, S. V., Corver, J., Lenches, E., Jones, C. T., Mukhopadhyay, S., Chipman, P. R., Strauss, E. G., Baker, T. S., and Strauss, J. H. (2002) Cell 108(5), 717-725
4. Kolaskar, A. S., and Kulkarni-Kale, U. (1999) Virology 261(1), 31-42
5. Modis, Y., Ogata, S., Clements, D., and Harrison, S. C. (2004) Nature 427(6972), 313-319
6. Konishi, E., Ajiro, N., Nukuzuma, C., Mason, P. W., and Kurane, I. (2003) Vaccine 21(25-26), 3675-3683
7. Butrapet, S., Kimura-Kuroda, J., Zhou, D. S., and Yasui, K. (1998) Am J Trop Med Hyg 58(4), 389-398
8. Cecilia, D., and Gould, E. A. (1991) Virology 181(1), 70-77
9. Lin, C. W., and Wu, S. C. (2003) J Virol 77(4), 2600-2606
10. Seif, S. A., Morita, K., Matsuo, S., Hasebe, F., and Igarashi, A. (1995) Vaccine 13(16), 1515-1521
11. Wu, S. C., Lian, W. C., Hsu, L. C., and Liau, M. Y. (1997) Virus Res 51(2), 173-181
12. Wu, K. P., Wu, C. W., Tsao, Y. P., Kuo, T. W., Lou, Y. C., Lin, C. W., Wu, S. C., and Cheng, J. W. (2003) J Biol Chem 278(46), 46007-46013
13. CHEN, Y., MAGUIRE, T., HILEMAN, R. E., RROMM, J. R., ESKO, J. D., LINHARDT, R. J., and MARKS, R. M. (1997) NATURE MEDCINE 3, 866-871
14. Liu, H., Chiou, S. S., and Chen, W. J. (2004) J Med Virol 72(4), 618-624
15. Marks, R. M., Lu, H., Sundaresan, R., Toida, T., Suzuki, A., Imanari, T., Hernaiz, M. J., and Linhardt, R. J. (2001) J Med Chem 44(13), 2178-2187
16. Su, C. M., Liao, C. L., Lee, Y. L., and Lin, Y. L. (2001) Virology 286(1), 206-215
17. Lee, E., and Lobigs, M. (2002) J Virol 76(10), 4901-4911
18. Chen, Y., Maguire, T., and Marks, R. M. (1996) J Virol 70(12), 8765-8772
19. Hung, J. J., Hsieh, M. T., Young, M. J., Kao, C. L., King, C. C., and Chang, W. (2004) J Virol 78(1), 378-388
20. Wu, S. C., Chiang, J. R., and Lin, C. W. (2004) Biomacromolecules 5(6), 2160-2164
21. Jaiswal, S., Khanna, N., and Swaminathan, S. (2004) Protein Expr Purif 33(1), 80-91