Skip to main content
SpringerLink
Log in

Modulation of enzymatic activity of dengue virus nonstructural protein NS3 nucleoside triphosphatase/helicase by poly(U)

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

The nonstructural protein 3 (NS3) appears to be the most promising target for anti-flavivirus therapy because of its multiple enzymatic activities that are indispensable for virus replication. NS3 of dengue virus type 2 (DEN2) is composed of two domains, a serine protease in the N-terminal domain (NS3pro) and RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase at the C-terminus (NS3h). NS3 plays an important role in viral replication and the coordinated regulation of all the catalytic activities in the full-length NS3 protein. In this study, a plasmid harboring the NS3 helicase domain (NS3h) was constructed by PCR. The 56.5 kDa NS3h protein was purified by metal-chelate affinity chromatography followed by renaturation, mediated by artificial chaperone-assisted refolding, which yielded the active helicase. NTPase activity was assayed with Malachite Green. The NTPase activity in the presence of poly(U) showed a higher turnover number (k cat) and a lower K m value than without poly(U). The activity increased approximately fourfold in the presence of polynucleotides. This indicates that NTPase activity of dengue NS3 can be stimulated by polynucleotides. A helicase assay based on internal fluorescence quenching was conducted using short internally quenched DNA oligonucleotides as substrates. Significant fluorescence signaling increase was observed in the absence of polynucleotides such as poly(U). No unwinding activity was observed with addition of poly(U). The approach we describe here is useful for the further characterization of substrate specificity and for the design of high-throughput assays aimed at discovery of inhibitors against NS3 NTPase/helicase activities.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. World Health Organization (2009) Geneva: World Health Organization and the Special Programme for Research and Training in Tropical Diseases (TDR), p. 147.

    Google Scholar 

  2. World Health Organization (2012) Dengue and Severe Dengue: Fact sheet number 117; available at http://www.who.int/mediacentre/factsheets/fs117/en/index.html (accessed February 22, 2013).

    Google Scholar 

  3. Chambers, T. J., Hahn, C. S., Galler, R., and Rice, C. M. (1990) Annu. Rev. Microbiol., 44, 649–688.

    Article  PubMed  CAS  Google Scholar 

  4. Lescara, J., Luo, D., Xub, T., Sampath, A., Lim, S. P., Canard, B., and Vasudeva, S. G. (2008) Antiviral Res., 80, 94–101.

    Article  Google Scholar 

  5. Gebhard, L. G., Kaufman, S. B., and Gamarnik, A. V. (2012) PLoS ONE, 7, e36244.

    Article  PubMed  CAS  Google Scholar 

  6. Sampath, A., and Padmanabhan, R. (2009) Antiviral Res., 81, 6–15.

    Article  PubMed  CAS  Google Scholar 

  7. Benarroch, D., Selisko, B., Locatelli, G. A., Maga, G., Romette, J. L., and Canarad, B. (2004) Virology, 328, 208–218.

    Article  PubMed  CAS  Google Scholar 

  8. Bartelma, G., and Padmanabhan, R. (2002) Virology, 299, 122–132.

    Article  PubMed  CAS  Google Scholar 

  9. Matusan, A. E., Pryor, M. J., Davidson, A. D., and Wright, P. J. (2001) J. Virol., 75, 9633–9643.

    Article  PubMed  CAS  Google Scholar 

  10. Ding, S. C., Kohlway, A. S., and Pyle, A. M. (2011) J. Virol., 85, 4343–4353.

    Article  PubMed  CAS  Google Scholar 

  11. Frick, D. N. (2006) in HCV Helicase: Structure, Function, and Inhibition (Tan, S. L., ed.) Chap. 6, Horizon Bioscience, Norfolk (UK) (http://www.ncbi.nlm.nih.gov/books/NBK1614/).

  12. Sikora, B., Chen, Y., Lichti, C. F., Harrison, M. K., Jennings, T. A., Tang, Y., Tackett, A. J., Jordan, J. B., Sakon, J., Cameron, C. E., and Raney, K. D. (2008) J. Biol. Chem., 283, 11516–11525.

    Article  PubMed  CAS  Google Scholar 

  13. Dumont, S., Cheng, W., Serebrov, V., Beran, R. K., Tinoco, I., Jr., Pyle, A. M., and Bustamante, C. (2006) Nature, 439, 105–108.

    Article  PubMed  CAS  Google Scholar 

  14. Serebrov, V., and Pyle, A. M. (2004) Nature, 430, 476–480.

    Article  PubMed  CAS  Google Scholar 

  15. Serebrov, V., Beran, R. K., and Pyle, A. M. (2009) J. Biol. Chem., 284, 2512–2521.

    Article  PubMed  CAS  Google Scholar 

  16. Cheng, W., Dumont, S., Tinoco, I., Jr., and Bustamante, C. (2007) Proc. Natl. Acad. Sci. USA, 104, 13954–13959.

    Article  PubMed  CAS  Google Scholar 

  17. Xu, T. A., Sampath, A., Chao, D., Wen, M., Nanao, P., Chene, Vasudevan, S. G., and Lescar, J. (2005) J. Virol., 79, 10278–10288.

    Article  PubMed  CAS  Google Scholar 

  18. Li, H., Clum, S., You, S., Ebner, K. E., and Padmanabhan, R. (1999) J. Virol., 73, 3108–3116.

    PubMed  CAS  Google Scholar 

  19. Beran, R. K., Serebrov, V., and Pyle, A. M. (2007) J. Biol. Chem., 282, 34913–34920.

    Article  PubMed  CAS  Google Scholar 

  20. Beran, R. K., and Pyle, A. M. (2008) J. Biol. Chem., 283, 29929–29937.

    Article  PubMed  CAS  Google Scholar 

  21. Rajagopal, V., Gurjar, M., Levin, M. K., and Patel, S. S. (2010) J. Biol. Chem., 285, 17821–17832.

    Article  PubMed  CAS  Google Scholar 

  22. Frick, D. N., Rypma, R. S., Lam, A. M., and Gu, B. (2004) J. Biol. Chem., 279, 1269–1280.

    Article  PubMed  CAS  Google Scholar 

  23. Mastrangelo, E., Milani, M., Bollati, M., Selisko, B., Peyrane, F., Pandini, V., Sorrentino, G., Canard, B., Konarev, P. V., Svergun, D. I., de Lamballerie, X., Coutard, B., Khromykh, A. A., and Bolognesi, M. (2007) J. Mol. Biol., 372, 444–455.

    Article  PubMed  CAS  Google Scholar 

  24. Lou, D., Xu, T., Hunke, C., Gruber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol., 82, 173–183.

    Article  Google Scholar 

  25. Banerjee, R., and Dasgupta, A. (2001) J. Virol., 75, 1708–1721.

    Article  PubMed  CAS  Google Scholar 

  26. Morgenstern, K. A., Lando, J. A., Hsiao, K., Lin, C., Gu, Y., Su, M. S., and Thomson, J. A. (1997) J. Virol., 71, 3767–3775.

    PubMed  CAS  Google Scholar 

  27. Wang, C. C., Huang, Z. S., Chiang, P. L., Chen, C. T., and Wu, H. N. (2009) FEBS Lett., 583, 691–696.

    Article  PubMed  CAS  Google Scholar 

  28. Bradford, M. M. (1996) Anal. Biochem., 72, 248–254.

    Article  Google Scholar 

  29. Lanzetta, P. A., Alvarez, L. J., Reinach, P. S., and Candia, O. A. (1979) Anal. Biochem., 100, 95–97.

    Article  PubMed  CAS  Google Scholar 

  30. Wardell, A. D., Errington, W., Ciaramella, G., Merson, J., and McGarvey, M. J. (1999) J. Gen. Virol., 80, 701–709.

    PubMed  CAS  Google Scholar 

  31. Anna, M. B., Krawczyk, M., Stankiewicz, A., Gozdek, A., Haenni, A., and Strokovskaya, L. (2004) FEBS, 567, 253–258.

    Article  Google Scholar 

  32. Machida, S., Ogawa, S., Xiaohua, S., Takaha, T., Fujii, K., and Hayashi, K. (2000) FEBS Lett., 486, 131–124.

    Article  PubMed  CAS  Google Scholar 

  33. Belon, C. A., and Frick, D. N. (2009) J. Mol. Biol., 388, 851–864.

    Article  PubMed  CAS  Google Scholar 

  34. Yon, C., Teramoto, T., Mueller, N., Phelan, J., Ganesh, V. K., Murthy, K. H. M., and Padmanabhan, R. (2005) J. Biol. Chem., 280, 27412–27419.

    Article  PubMed  CAS  Google Scholar 

  35. Tai, C. L., Chi, W. K., Chen, D. S., and Hwang, L. H. (1996) J. Virol., 70, 8477–8484.

    PubMed  CAS  Google Scholar 

  36. Suzich, J. A., Tamura, J. K., Palmer-Hill, F., Warrener, P., Grakoui, A., Rice, C. M., Feinstone, S. M., and Collett, M. S. (1993) J. Virol., 67, 6152–6158.

    PubMed  CAS  Google Scholar 

  37. Maga, G., Gemma, S., Fattorusso, C., Locatelle, G. A., Butini, S., Persico, M., Kukreja, G., Romano, M. P., Chiasserini, L., Savini, L., Novellino, E., Nacci, V., Spadari, S., and Campiani, G. (2005) Biochemistry, 44, 9637–9644.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory of Molecular and Cellular Microbiology, Institute of Molecular Biosciences, Mahidol University, Salaya, 73170, Thailand

    M. Junaid, C. Angsuthanasombat & G. Katzenmeier

  2. Department of Pharmacy, University of Malakand, K. P. K, Malakand, 18550, Pakistan

    M. Junaid

  3. Department of Pharmaceutical Biosciences, Division of Pharmacology, Uppsala University, Uppsala, 75124, Sweden

    J. E. S. Wikberg

  4. Department of Pharmacology, Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Khyber Pakhtunkhwa, 25000, Pakistan

    N. Ali

Authors
  1. M. Junaid
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C. Angsuthanasombat
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. E. S. Wikberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. N. Ali
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. G. Katzenmeier
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G. Katzenmeier.

Additional information

Published in Russian in Biokhimiya, 2013, Vol. 78, No. 8, pp. 1178–1186.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM13-082, July 14, 2013.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Junaid, M., Angsuthanasombat, C., Wikberg, J.E.S. et al. Modulation of enzymatic activity of dengue virus nonstructural protein NS3 nucleoside triphosphatase/helicase by poly(U). Biochemistry Moscow 78, 925–932 (2013). https://doi.org/10.1134/S0006297913080105

Download citation

  • Received:

  • Revised:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297913080105

Key words

Search

Navigation