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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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In Vitro Determination of Dengue Virus Type 2 NS2B-NS3 Protease Activity with Fluorescent Peptide Substrates

  • Khumthong, Rabuesak (Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus) ;
  • Angsuthanasombat, Chanan (Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus) ;
  • Panyim, Sakol (Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus) ;
  • Katzenmeier, Gerd (Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus)
  • Published : 2002.03.31
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Abstract

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2B-NS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.

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References

  1. Arias, C. F., Preugschat, F. and Strauss, J. H. (1993) Dengue 2 virus NS2B and NS3 fonn a stable complex that can cleave NS3 within the helicase domain. Virology 193, 888-899. https://doi.org/10.1006/viro.1993.1198
  2. Bazan, J. F. and Fletterick, R. J. (1989) Detection of a trypsin-like serine protease domain in flaviviruses and pestiviruses. Virology 171, 637-639. https://doi.org/10.1016/0042-6822(89)90639-9
  3. Bhamarapravati, N. and Sutee, Y. (2000) Live attenuated tetravalent dengue vaccine. Vaccine 18, 44-47. https://doi.org/10.1016/S0264-410X(00)00040-2
  4. Brinkworth, R. I., Fairlie, D. P., Leung, D. and Young, P. R. (1999) Homology model of the dengue 2 virus NS3 protease: putative interactions with both substrate and NS2B cofactor. J. Gen. Virol. 80, 1167 -1177 https://doi.org/10.1099/0022-1317-80-5-1167
  5. Chambers, T. J., Hahn, C. S., Galler, R. and Rice, C. M. (1990) F1avivirus genome organization, expression and replication. Annu. Rev. Microbiol. 44, 649-688. https://doi.org/10.1146/annurev.mi.44.100190.003245
  6. Chambers, T. J., Nestorowitz, A. and Rice, C. M. (1995) Mutagenesis of the yellow fever virus NS2B/3 cleavage site: determinants of cleavage site specificity and effects on polyprotein processing and viral replication. J. Virol. 69, 1600-1605.
  7. Champreda, V., Khumthong, R., Subsin, B., Angsuthanasombat, C., Panyim, S. and Katzenmeier, G. (2000) The two-component protease NS2B-NS3 of dengue virus type 2: cloning, expression in Escherichia coli and purification of the NS2B, NS3(pro) and NS2B-NS3 proteins. J. Biochem. Mol. BioI. 33, 294-299.
  8. Falgout, B. and Markoff, L. (1995) Evidence that flavivirus NS1- NS2A cleavage is mediated by a membrane-bound host protease in the endoplasmic reticulum. J. Virol. 69, 7232-7234.
  9. Falgout, B., Miller, R. H. and 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.
  10. Falgout, B., Pethel, M., Zhang, Y. M. and 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.
  11. Gubler, D. J. and Clark, G. G. (1995) Dengue/dengue hemorrhagic fever: the emergence of a global health problem. Emerg. lnfect. Dis. 1, 55-57. https://doi.org/10.3201/eid0102.950204
  12. Huang, C. Y., Butrapet, S., Pierro, D. J., Chang, G. J., Hunt, A. R., Bhamarapravati, N., Gubler, D. J. and Kinney, R. M. (2000) Chimeric dengue type 2 (vaccine strain PDK-53)/ dengue type 1 virus as a potential candidate dengue type 1 virus vaccine. J. Virol. 74, 3020-3028. https://doi.org/10.1128/JVI.74.7.3020-3028.2000
  13. Kadare, G. and Haenni, A. L. (1997) Virus-encoded RNA helicases. J. Virol. 71, 2583-2590.
  14. Kakiuchi, N., Komoda, Y., Hijikata, M. and Shimotohno K. (1997) Cleavage activity of hepatitis C virus serine protease. J. Biochem. 122,749-755. https://doi.org/10.1093/oxfordjournals.jbchem.a021819
  15. Kim, J. L., Morgenstern, K. A., Lin, C., Fox, T., Dwyer, M. D., Landro, J. A., Chambers, S. P., Markland, W., Lepre, C. A., OMalley, E. T., Harbeson, S. L., Rice, C. M., Murcko, M. A., Caron, P. R. and Thomson, J. A. (1996) Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell 87, 343-355. https://doi.org/10.1016/S0092-8674(00)81351-3
  16. Kinney, R. M., Butrapet, S., Chang, G. J., Tsuchiya, K. R., Roehrig, J. T., Bhamarapravati, N. and Gubler, D. J. (1997) Construction of infectious cDNA clones for dengue 2 virus strain 16681 and its attenuated vaccine derivative, strain PDK- 53. Virology 230, 300-308. https://doi.org/10.1006/viro.1997.8500
  17. Li, H., Clum, S., You, S., Ebner, K. E. and Padmanabhan, R.(1999) The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids. J. Virol. 73, 3108-3116.
  18. Lin, C., Amberg, S. M., Chambers, T. J. and 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.
  19. Lobigs, M. (1993) Flavivirus premembrane protein cleavage and spike heterodimer secretion require the function of the viral proteinase NS3. Proc. Natl. Acad. Sci. USA 90, 6218-6222. https://doi.org/10.1073/pnas.90.13.6218
  20. Markoff, L. (1989) In vitro processing of dengue virus structural proteins: cleavage of the pre-membrane protein. J. Virol. 63, 3345-3352.
  21. Monath, T. P. (1994) Dengue: the risk to developed and developing countries. Proc. Natl. Acad. Sci. USA 91, 2395-2400. https://doi.org/10.1073/pnas.91.7.2395
  22. Murthy, H. M., Clum, S. and Padmanabhan, R. (1999) Dengue virus NS3 serine protease. J. BioI. Chem. 274, 5573-5580. https://doi.org/10.1074/jbc.274.9.5573
  23. Murthy, H. M., Judge, K., DeLucas, L. and Padmanabhan, R. (2000) Crystal structure of dengue virus NS3 protease in complex with a Bowman-Birk inhibitor: implications for flaviviral polyprotein processing and drug design. J. Mol. Biol. 301, 759-767. https://doi.org/10.1006/jmbi.2000.3924
  24. Nowak, T., Farber, T. M., Wengler, G. and Wengler, G. (1989) Analyses of the terminal sequences of West Nile virus structural proteins and of the in vitro translation of these proteins allow the proposal of a complete scheme of the proteolytic cleavages involved in their synthesis. Virology 169, 365-376. https://doi.org/10.1016/0042-6822(89)90162-1
  25. Rigau-Perez, J. G., Clark, G. G., Gubler, D. J., Reiter, P., Sanders, E. J. and Vomdam, A. V. (1998) Dengue and dengue hemorrhagic fever. Lancet 352, 971-976. https://doi.org/10.1016/S0140-6736(97)12483-7
  26. Seybert, A., Ziebuhr, J. and Siddel, S. G. (1997) Expression and characterization of a recombinant murine coronovirus 3C-like proteinase. J. Gen. Virol. 78, 71-75. https://doi.org/10.1099/0022-1317-78-1-71
  27. Teo, K. F. and Wright, P. J. (1997) Internal proteolysis of the NS3 protein specified by dengue virus type 2. J. Virol. 72, 624-632.
  28. Wang, Q. M., Sommergruber, W. and Johnson, R. B. (1997) Cleavage specificity of human rhinovirus-2 2A protease for peptide substrates. Biochem Biophys. Res. Commun. 235, 562-566. https://doi.org/10.1006/bbrc.1997.6830
  29. Yusof, R., Clum, S., Wetzel, M., Krishna Murthy, H. M. and Padmanabhan, R. (2000) Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro. J. BioI. Chem. 275, 9963-9969. https://doi.org/10.1074/jbc.275.14.9963

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