Advertisement

Molecular Biology

, Volume 44, Issue 6, pp 931–938 | Cite as

Interactions between the hepatitis C virus protein NS3 and polymethylene derivatives of nucleic bases

  • A. V. Mukovnya
  • V. V. Komissarov
  • A. M. Kritsyn
  • V. A. Mitkevich
  • V. L. Tunitskaya
  • S. N. KochetkovEmail author
Structural-Functional Analysis of Biopolymers and Their Complexes
  • 33 Downloads

Abstract

Nonstructural protein 3 (NS3) of hepatitis C virus plays a key role in the functioning of the virus. NS3 displays three enzymatic activities, namely, protease activity associated with the N-terminal domain, coupled nucleoside triphosphotase (NTPase), and helicase activities, localized to the C-terminal domain. In this work, we studied the effects of various polymethylene derivatives of nucleic bases on the NTPase (by the example of ATPase) and helicase activities of NS3. It was demonstrated that some tested compounds inhibited NS3 helicase activity; however, a considerable part of the compounds activated the NTPase activity of NS3 and several other proteins displaying NTPase or selective ATPase activity. Such ATPase activators have not been earlier described, suggesting an unusual activation mechanism. The activation ability of the tested compounds depended on the ratio of substrate (ATP) and activator concentrations, and reached its maximum at a 1000-fold excess of the substrate. A mechanism of ATPase activation was proposed to explain the observed effects.

Keywords

hepatitis C virus nonstructural protein NS3 ATPase activity helicase activity activation inhibition polymethylene derivatives of nucleic bases 

Abbreviations

ADPNP

adenylyl imidodiphosphate

HCV

hepatitis C virus

NS3

HCV nonstructural protein 3

NS3FL

full-length NS3

NS3hel

NS3 helicase domain

IPTG

isopropyl thiogalactoside

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Bartenschlager R., Lohmann V. 2000. Replication of hepatitis C virus. J. Gen. Virol.. 81, 1631–1648.PubMedGoogle Scholar
  2. 2.
    Zhang C., Cai Z, Kim Y.-C., Kumar R., Yuan F, Shi P.-Y., Kao C., Luo G. 2005. Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase. J. Virol. 79, 8687–8697.CrossRefPubMedGoogle Scholar
  3. 3.
    Global surveillance and control of hepatitis C. Report of a WHO Consultation organized in collaboration with the Viral Hepatitis Prevention Board, Antwerp, Belgium. 1999. J. Viral Hepat. 6, 35–47.Google Scholar
  4. 4.
    Lindenbach B.D., Rice C.M. 2001. Flaviviridae: The virusis and their replication. In: Fundamental Virology. Eds Knipe D.M., Howley P.M. Philadelphia: SE Straus Lappincott Williams and Wilkins, 589–639.Google Scholar
  5. 5.
    Bukh J., Purcell R.H., Miller R.H. 1992. Sequence analysis of the 5′ noncoding region of hepatitis C virus. Proc. Natl. Acad. Sci. USA. 89, 4942–4946.CrossRefPubMedGoogle Scholar
  6. 6.
    Pestova T.V., Kolupaeva V.G., Lomakin I.B., Pilipenko E.V., Shatsky I.N., Agol V.I., Hellen C.U. 2001. Molecular mechanisms of translation initiation in eukaryotes. Proc. Natl. Acad. Sci. USA. 98, 7029–7036.CrossRefPubMedGoogle Scholar
  7. 7.
    De Francesco R., Tomei L., Altamura S., Summa V., Migliaccio G. 2003. Approaching a new era for hepatitis C virus therapy: inhibitors of the NS3-4A serine protease and the NS5B RNA-dependent RNA polymerase. Antiviral Res. 58, 1–16.CrossRefPubMedGoogle Scholar
  8. 8.
    Mukovnya A.V., Tunitskaya V.L., Khandazhinskaya A.L., Golubeva N.A., Zakirova N.F., Ivanov A.V., Kukhanova M.K., Kochetkov S.N. 2008. Hepatitis C virus helicase/NTPase: Effective expression system and new inhibitors. Biokhimiya. 73, 822–832.Google Scholar
  9. 9.
    Paeshuyse J., Vliegen I., Coelmont L., Leyssen P., Tabarrini O., Herdewijn P., Mittendorfer H., Easmon J., Cecchetti V., Bartenschlager R., Puerstinger G., Neyts J. 2008. Comparative in vitro anti-hepatitis C virus activities of a selected series of polymerase, protease, and helicase inhibitors. Antimicrob. Agents Chemotherapy. 52, 3433–3437.CrossRefGoogle Scholar
  10. 10.
    Borowski P., Deinert J., Schalinski S., Bretner M., Ginalski K., Kulikowski T., Shugar D. 2003. Halogenated benzimidazoles and benzotriazoles as inhibitors of the NTPase/helicase activities of hepatitis C and related viruses. Eur. J. Biochem. 270, 1645–1653.CrossRefPubMedGoogle Scholar
  11. 11.
    Borowski P., Lang M., Haag A., Schmitz H., Choe J., Chen H.-M., Ramachandra S. 2002. Hosmane characterization of imidazo[4.5-d]pyridazine nucleosides as modulators of unwinding reaction mediated by West Nile virus nucleoside triphosphatase/helicase: Evidence for activity on the level of substrate and/or enzyme. Antimicrob. Agents Chemother. 1231–1239.Google Scholar
  12. 12.
    Ivanov A.V., Korovina A.N., Tunitskaya V.L., Kostyuk D.A., Rechinsky V.O., Kukhanova M.K., Kochetkov S.N. 2006. Development of the system ensuring a high-level expression of hepatitis C virus nonstructural NS5B and NS5A proteins. Protein Expr. Purif. 48, 14–23.CrossRefPubMedGoogle Scholar
  13. 13.
    Sambrook J., Fritsch E.F., Maniatis T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Lab. Press.Google Scholar
  14. 14.
    Mitkevich V.A,, Kononenko A.V., Petrushanko I.Y., Yanvarev D.V., Makarov A.A., Kisselev L.L. 2006. Termination of translation in eukaryotes is mediated by the quaternary eRF1*eRF3*GTP*Mg2+ complex. The biological roles of eRF3 and prokaryotic RF3 are profoundly distinct. Nucleic Acids Res. 34, 3947–3954.CrossRefPubMedGoogle Scholar
  15. 15.
    Makinskii A.A., Kritsyn A.M., Ul’yanova E.A., Zakharova O.D., Nevinskii G.A. 2000. Polymethylene derivatives of nucleic bases with ω-functional groups: Pyrimidine derivatives. Bioorg. Khim. 26, 735–742.PubMedGoogle Scholar
  16. 16.
    Kritsyn A.M., Komissarov V.V. 2005. Polymethylene derivatives of nucleic bases with ω-functional groups: 5. Pyrimidine- and purine-containing γ-butyrophenones. Bioorg. Khim. 31, 609–615.PubMedGoogle Scholar
  17. 17.
    Summa V., Petrocchi A., Matassa V.G., Taliani M., Laufer R., De Francesco R., Altamura S., Pace P. 2004. HCV NS5b RNA-dependent RNA polymerase inhibitors: From alpha,gamma-diketoacids to 4.5-dihydroxy-pyrimidine- or 3-methyl-5-hydroxypyrimidinonecarboxylic acids. Design and synthesis. J. Med. Chem. 47, 5336–5339.CrossRefPubMedGoogle Scholar
  18. 18.
    Biswal B.K., Cherney M.M., Wang M., Chan L., Yannopoulos C.G., Bilimoria D., Nicolas O., Bedard J., James M.N.G. 2005. Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus reveal two conformations and suggest mechanisms of inhibition by non-nucleoside inhibitors. J. Biol. Chem. 280, 18202–18210.CrossRefPubMedGoogle Scholar
  19. 19.
    Summa V., Petrocchi A., Pace P., Matassa V.G., De Francesco R., Altamura S., Tomei L., Koch U., Neuner P. 2004. Discovery of alpha,gamma-diketo acids as potent selective and reversible inhibitors of hepatitis C virus NS5b RNA-dependent RNA polymerase. J. Med. Chem. 47, 14–17.CrossRefPubMedGoogle Scholar
  20. 20.
    Wai J.S., Egbertson M.S., Payne L.S., Fisher T.E., Embrey M.W., Tran L.O., Melamed J.Y., Langford H.M., Guare J.P., Jr., Zhuang L., Grey V.E., Vacca J.P., Holloway M.K., Naylor-Olsen A.M., Hazuda D.J., Felock P.J., Wolfe A.L., Stillmock K.A., Schleif W.A., Gabryelski L.J., Young S.D. 2000. 4-Aryl-2.4-dioxobutanoic acid inhibitors of HIV-1 integrase and viral replication in cells. J. Med. Chem. 43, 4923–4926.CrossRefPubMedGoogle Scholar
  21. 21.
    Grobler J.A., Stillmock K., Hu B., Witmer M., Felock P., Espeseth A.S., Wolfe A., Egbertson M., Bourgeois M., Melamed J., Wai J.S., Young S., Vacca J., Hazuda D.J. 2002. Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes. Proc. Natl. Acad. Sci. USA. 99, 6661–6666.CrossRefPubMedGoogle Scholar
  22. 22.
    Long Y.-Q., Jiang X.-H., Dayam R., Sanchez T., Shoemaker R., Sei S., Neamati N. 2004. Rational design and synthesis of novel dimeric diketoacid-containing inhibitors of HIV-1 integrase: Implication for binding to two metal ions on the active site of integrase. J. Med. Chem. 47, 2561–2573.CrossRefPubMedGoogle Scholar
  23. 23.
    Kel’vin A. V. 2003. Recent advances in the synthesis of 1.3-diketones. Curr. Org. Chem. 7, 1691–1711.CrossRefGoogle Scholar
  24. 24.
    Komissarov V.V., Panova N.G., Kritsyn A.M. 2008. [8-(2-oxocyclohexyl)-8-oxooktyl]pyrimidines: Potential inhibitors of pyrimidine phosphorylases. Bioorg. Khim. 34, 75–82.PubMedGoogle Scholar
  25. 25.
    Komissarov, V.V., Volgareva, G.M., Ol’shanskaya, Ya.S., Chernyshova, M.E., Zavalishina L.E., Frank G.A., Shtil’ A.A., Kritsyn A.M. 2009. Polymethylene derivatives of nucleic bases with ω-functional groups: 7. Cytotoxicity in the series of N-(2-oxocyclohexyl)-ω-oxoalkyl-substituted purines and pyrimidines. Bioorg. Khim. 35, 84–94.PubMedGoogle Scholar
  26. 26.
    Vorob’ev Yu.N. 1984. A method of computing conformations of lagre nucleic acid fragments. V. Modified TΨC loop of phenylalanine tRNA. Mol. Biol. (Moscow) 18, 756–765.Google Scholar
  27. 27.
    Wardell A.D., Errington W., Ciaramella G., Merson J., McGarvey M.J. 1999. Characterization and mutational analysis of the helicase and NTPase activities of hepatitis C virus full-length NS3 protein. J. Gen. Virol. 80, 701–709.PubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2010

Authors and Affiliations

  • A. V. Mukovnya
    • 1
  • V. V. Komissarov
    • 1
  • A. M. Kritsyn
    • 1
  • V. A. Mitkevich
    • 1
  • V. L. Tunitskaya
    • 1
  • S. N. Kochetkov
    • 1
    Email author
  1. 1.Engelhardt Institute of Molecular BiologyRussian Academy of SciencesMoscowRussia

Personalised recommendations