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Abstract

West Nile virus (WNV) is an emerging mosquito-borne flavivirus that causes neuronal damage in the absence of treatment. In many flaviviruses, including WNV, the NS2B cofactor promotes the productive folding and the functional activity of the two-component NS3 (pro)teinase. Based on an analysis of the NS2B–NS3pro structure, we hypothesized that the G residue and the negatively charged patch DDD of NS2B were part of an important configuration required for NS2B–NS3pro activity. Our experimental data confirmed that G and DDD substitution for S and AAA, respectively, inactivated NS2B–NS3pro. An additional D42G mutant, which we designed as a control, had no dramatic effect on either the catalytic activity or self-proteolysis of NS2B–NS3pro. Because of the significant level of homology in flaviviral NS2B–NS3pro, our results will be useful for the development of specific allosteric inhibitors designed to interfere with the productive interactions of NS2B with NS3pro.

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2008-03-01
2019-10-19
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References

  1. Aleshin, A. E., Shiryaev, S. A., Strongin, A. Y. & Liddington, R. C. ( 2007; ). Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold. Protein Sci 16, 795–806.[CrossRef]
    [Google Scholar]
  2. Beasley, D. W. ( 2005; ). Recent advances in the molecular biology of West Nile virus. Curr Mol Med 5, 835–850.[CrossRef]
    [Google Scholar]
  3. Bessaud, M., Grard, G., Peyrefitte, C. N., Pastorino, B., Rolland, D., Charrel, R. N., de Lamballerie, X. & Tolou, H. J. ( 2005; ). Identification and enzymatic characterization of NS2B–NS3 protease of Alkhurma virus, a class-4 flavivirus. Virus Res 107, 57–62.[CrossRef]
    [Google Scholar]
  4. Cahour, A., Falgout, B. & Lai, C. J. ( 1992; ). Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B–NS3, whereas NS4A/NS4B may be processed by a cellular protease. J Virol 66, 1535–1542.
    [Google Scholar]
  5. Chambers, T. J., Nestorowicz, A., Amberg, S. M. & Rice, C. M. ( 1993; ). Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B–NS3 complex formation, and viral replication. J Virol 67, 6797–6807.
    [Google Scholar]
  6. Chambers, T. J., Droll, D. A., Tang, Y., Liang, Y., Ganesh, V. K., Murthy, K. H. & Nickells, M. ( 2005; ). Yellow fever virus NS2B–NS3 protease: characterization of charged-to-alanine mutant and revertant viruses and analysis of polyprotein-cleavage activities. J Gen Virol 86, 1403–1413.[CrossRef]
    [Google Scholar]
  7. Chappell, K. J., Stoermer, M. J., Fairlie, D. P. & Young, P. R. ( 2006; ). Insights to substrate binding and processing by West Nile virus NS3 protease through combined modeling, protease mutagenesis, and kinetic studies. J Biol Chem 281, 38448–38458.[CrossRef]
    [Google Scholar]
  8. Chappell, K. J., Stoermer, M. J., Fairlie, D. P. & Young, P. R. ( 2007; ). Generation and characterization of proteolytically active and highly stable truncated and full-length recombinant West Nile virus NS3. Protein Expr Purif 53, 87–96.[CrossRef]
    [Google Scholar]
  9. Droll, D. A., Krishna Murthy, H. M. & Chambers, T. J. ( 2000; ). Yellow fever virus NS2B–NS3 protease: charged-to-alanine mutagenesis and deletion analysis define regions important for protease complex formation and function. Virology 275, 335–347.[CrossRef]
    [Google Scholar]
  10. Erbel, P., Schiering, N., D'Arcy, A., Renatus, M., Kroemer, M., Lim, S. P., Yin, Z., Keller, T. H., Vasudevan, S. G. & Hommel, U. ( 2006; ). Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus. Nat Struct Mol Biol 13, 372–373.[CrossRef]
    [Google Scholar]
  11. Falgout, B., Pethel, M., Zhang, Y. M. & Lai, C. J. ( 1991; ). Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol 65, 2467–2475.
    [Google Scholar]
  12. Falgout, B., Miller, R. H. & Lai, C. J. ( 1993; ). Deletion analysis of dengue virus type 4 nonstructural protein NS2B: identification of a domain required for NS2B–NS3 protease activity. J Virol 67, 2034–2042.
    [Google Scholar]
  13. Knox, J. E., Ma, N. L., Yin, Z., Patel, S. J., Wang, W. L., Chan, W. L., Ranga Rao, K. R., Wang, G., Ngew, X. & other authors ( 2006; ). Peptide inhibitors of West Nile NS3 protease: SAR study of tetrapeptide aldehyde inhibitors. J Med Chem 49, 6585–6590.[CrossRef]
    [Google Scholar]
  14. Leung, D., Schroder, K., White, H., Fang, N. X., Stoermer, M. J., Abbenante, G., Martin, J. L., Young, P. R. & Fairlie, D. P. ( 2001; ). Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors. J Biol Chem 276, 45762–45771.[CrossRef]
    [Google Scholar]
  15. Lin, C., Amberg, S. M., Chambers, T. J. & Rice, C. M. ( 1993; ). Cleavage at a novel site in the NS4A region by the yellow fever virus NS2B–3 proteinase is a prerequisite for processing at the downstream 4A/4B signalase site. J Virol 67, 2327–2335.
    [Google Scholar]
  16. Lin, C. W., Huang, H. D., Shiu, S. Y., Chen, W. J., Tsai, M. H., Huang, S. H., Wan, L. & Lin, Y. J. ( 2007; ). Functional determinants of NS2B for activation of Japanese encephalitis virus NS3 protease. Virus Res 127, 88–94.[CrossRef]
    [Google Scholar]
  17. Lohr, K., Knox, J. E., Phong, W. Y., Ma, N. L., Yin, Z., Sampath, A., Patel, S. J., Wang, W. L., Chan, W. L. & other authors ( 2007; ). Yellow fever virus NS3 protease: peptide-inhibition studies. J Gen Virol 88, 2223–2227.[CrossRef]
    [Google Scholar]
  18. Madden, K. ( 2003; ). West Nile virus infection and its neurological manifestations. Clin Med Res 1, 145–150.[CrossRef]
    [Google Scholar]
  19. Mukhopadhyay, S., Kuhn, R. J. & Rossmann, M. G. ( 2005; ). A structural perspective of the flavivirus life cycle. Nat Rev Microbiol 3, 13–22.[CrossRef]
    [Google Scholar]
  20. Nall, T. A., Chappell, K. J., Stoermer, M. J., Fang, N. X., Tyndall, J. D., Young, P. R. & Fairlie, D. P. ( 2004; ). Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease. J Biol Chem 279, 48535–48542.[CrossRef]
    [Google Scholar]
  21. Niyomrattanakit, P., Winoyanuwattikun, P., Chanprapaph, S., Angsuthanasombat, C., Panyim, S. & Katzenmeier, G. ( 2004; ). Identification of residues in the dengue virus type 2 NS2B cofactor that are critical for NS3 protease activation. J Virol 78, 13708–13716.[CrossRef]
    [Google Scholar]
  22. Pastorino, B. A., Peyrefitte, C. N., Grandadam, M., Thill, M. C., Tolou, H. J. & Bessaud, M. ( 2006; ). Mutagenesis analysis of the NS2B determinants of the Alkhurma virus NS2B–NS3 protease activation. J Gen Virol 87, 3279–3283.[CrossRef]
    [Google Scholar]
  23. Preugschat, F. & Strauss, J. H. ( 1991; ). Processing of nonstructural proteins NS4A and NS4B of dengue 2 virus in vitro and in vivo. Virology 185, 689–697.[CrossRef]
    [Google Scholar]
  24. Shiryaev, S. A., Ratnikov, B. I., Chekanov, A. V., Sikora, S., Rozanov, D. V., Godzik, A., Wang, J., Smith, J. W., Huang, Z. & other authors ( 2006; ). Cleavage targets and the d-arginine-based inhibitors of the West Nile virus NS3 processing proteinase. Biochem J 393, 503–511.[CrossRef]
    [Google Scholar]
  25. Shiryaev, S. A., Aleshin, A. E., Ratnikov, B. I., Smith, J. W., Liddington, R. C. & Strongin, A. Y. ( 2007; ). Expression and purification of a two-component flaviviral proteinase resistant to autocleavage at the NS2B–NS3 junction region. Protein Expr Purif 52, 334–339.[CrossRef]
    [Google Scholar]
  26. van der Meulen, K. M., Pensaert, M. B. & Nauwynck, H. J. ( 2005; ). West Nile virus in the vertebrate world. Arch Virol 150, 637–657.[CrossRef]
    [Google Scholar]
  27. Wang, T., Town, T., Alexopoulou, L., Anderson, J. F., Fikrig, E. & Flavell, R. A. ( 2004; ). Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis. Nat Med 10, 1366–1373.[CrossRef]
    [Google Scholar]
  28. Wu, C. F., Wang, S. H., Sun, C. M., Hu, S. T. & Syu, W. J. ( 2003; ). Activation of dengue protease autocleavage at the NS2B–NS3 junction by recombinant NS3 and GST-NS2B fusion proteins. J Virol Methods 114, 45–54.[CrossRef]
    [Google Scholar]
  29. Yin, Z., Patel, S. J., Wang, W. L., Chan, W. L., Ranga Rao, K. R., Wang, G., Ngew, X., Patel, V., Beer, D. & other authors ( 2006; ). Peptide inhibitors of dengue virus NS3 protease. Part 2: SAR study of tetrapeptide aldehyde inhibitors. Bioorg Med Chem Lett 16, 40–43.[CrossRef]
    [Google Scholar]
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