1887

Abstract

The hepatitis C virus non-structural protein 3 (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion. In this study, an N-terminal hexahistidine-tagged full-length NS3 polypeptide was expressed in and purified to homogeneity by conventional chromatography. Detailed characterization of the helicase activity of NS3 is presented with regard to its binding and strand release activities on different RNA substrates. On RNA double-hybrid substrates, the enzyme was shown to perform unwinding activity starting from an internal ssRNA region of at least 3 nt and moving along the duplex in a 3′ to 5′ direction. In addition, data are presented suggesting that binding to ATP reduces the affinity of NS3 for ssRNA and increases its affinity for duplex RNA. Furthermore, we have ascertained the capacity of NS3 to specifically interact with and resolve the stem–loop RNA structure (SL I) within the 3′-terminal 46 bases of the viral genome. Finally, our analysis of NS3 processive unwinding under single cycle conditions by addition of heparin in both helicase and RNA-stimulated ATPase assays led to two conclusions: (i) NS3-associated helicase acts processively; (ii) most of the NS3 RNA-stimulated ATPase activity may not be directly coupled to translocation of the enzyme along the substrate RNA molecule.

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2000-05-01
2019-10-13
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References

  1. Bird, L. E., Subramanya, H. S. & Wingley, D. B. ( 1998; ). Helicases: a unifying structural theme? Current Opinion in Structural Biology 8, 14-18.[CrossRef]
    [Google Scholar]
  2. Blight, K. J. & Rice, C. M. ( 1997; ). Secondary structure determination of the conserved 98-base sequence at the 3′ terminus of hepatitis C virus genome RNA. Journal of Virology 71, 7345-7352.
    [Google Scholar]
  3. Boehmer, P. E. ( 1998; ). The herpes simplex virus type-1 single-strand DNA-binding protein, ICP8, increases the processivity of the UL9 protein DNA helicase. Journal of Biological Chemistry 273, 2676-2683.[CrossRef]
    [Google Scholar]
  4. Chen, C.-J., Kuo, M.-D., Chien, L.-J., Hsu, S.-L., Wang, Y.-M. & Lin, J.-H. ( 1997; ). RNA–protein interactions: involvement of NS3, NS5, and 3′ noncoding regions of Japanese encephalitis virus genomic RNA. Journal of Virology 71, 3466-3473.
    [Google Scholar]
  5. Cheng, J.-C., Chang, M.-F. & Chang, S. C. ( 1999; ). Specific interaction between the hepatitis C virus NS5B RNA polymerase and the 3′ end of the viral RNA. Journal of Virology 73, 7044-7049.
    [Google Scholar]
  6. Cho, H.-S., Ha, N.-C., Kang, L.-W., Chung, K. M., Back, S. H., Jang, S. K. & Oh, B.-H. ( 1998; ). Crystal structure of RNA helicase from genotype 1b hepatitis C virus. Journal of Biological Chemistry 273, 15045-15052.[CrossRef]
    [Google Scholar]
  7. Clarke, B. ( 1997; ). Molecular virology of hepatitis C virus. Journal of General Virology 78, 2397-2410.
    [Google Scholar]
  8. Cui, T., Sugrue, R. J., Xu, Q., Lee, A. K. W., Chan, Y.-C. & Fu, J. ( 1998; ). Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein. Virology 246, 409-417.[CrossRef]
    [Google Scholar]
  9. De Francesco, R., Pessi, A. & Steinkühler, C. ( 1998; ). The hepatitis C virus NS3 proteinase: structure and function of a zinc-containing serine proteinase. In Therapies for Viral Hepatitis, pp. 235-245. Edited by R. F. Schinazi, J.-P. Sommadossi & H. C. Thomas. London: International Medical Press.
  10. Dong, F., Weitzel, S. E. & von Hippel, P. H. ( 1996; ). A coupled complex of T4 DNA replication helicase (gp41) and polymerase (gp43) can perform rapid and processive DNA strand-displacement synthesis. Proceedings of the National Academy of Sciences, USA 93, 14456-14461.[CrossRef]
    [Google Scholar]
  11. Gallinari, P., Brennan, D., Nardi, C., Brunetti, M., Tomei, L., Steinkühler, C. & De Francesco, R. ( 1998; ). Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus. Journal of Virology 72, 6758-6769.
    [Google Scholar]
  12. Gallinari, P., Paolini, C., Brennan, D., Nardi, C., Steinkühler, C. & De Francesco, R. ( 1999; ). Modulation of hepatitis C virus NS3 protease and helicase activities through the interaction with NS4A. Biochemistry 38, 5620-5632.[CrossRef]
    [Google Scholar]
  13. Gorbalenya, A. E. & Koonin, E. V. ( 1993; ). Helicases: amino acid sequence comparison and structure–function relationship. Current Opinion in Structural Biology 3, 419-429.[CrossRef]
    [Google Scholar]
  14. Gwack, Y., Wook, D., Han, J. H. & Choe, J. ( 1995; ). NTPase activity of hepatitis C virus NS3 protein expressed in insect cells. Molecular Cell 5, 171-175.
    [Google Scholar]
  15. Gwack, Y., Kim, D. W., Han, J. H. & Choe, J. ( 1996; ). Characterization of RNA binding activity and RNA helicase activity of the hepatitis C virus NS3 protein. Biochemical and Biophysical Research Communications 225, 654-659.[CrossRef]
    [Google Scholar]
  16. Houghton, M. ( 1996; ). Hepatitis C viruses. In Fields Virology, pp. 1035-1058. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. New York: Raven Press.
  17. Ito, T. & Lai, M. C. ( 1997; ). Determination of the secondary structure of and cellular protein binding to the 3′-untranslated region of the hepatitis C virus RNA genome. Journal of Virology 71, 8698-8706.
    [Google Scholar]
  18. Jin, L. & Peterson, D. L. ( 1995; ). Expression, isolation, and characterization of the hepatitis C virus ATPase/RNA helicase. Archives of Biochemistry & Biophysics 323, 47-53.[CrossRef]
    [Google Scholar]
  19. Kanai, A., Tanabe, K. & Kohara, M. ( 1995; ). Poly(U) binding activity of hepatitis C virus NS3 protein, a putative RNA helicase. FEBS Letters 376, 221-224.[CrossRef]
    [Google Scholar]
  20. Kim, D. W., Gwack, Y., Han, J. H. & Choe, J. ( 1995; ). C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochemical and Biophysical Research Communications 215, 160-166.[CrossRef]
    [Google Scholar]
  21. Kim, D. W., Kim, J., Gwack, Y., Han, J. H. & Choe, J. ( 1997; ). Mutational analysis of the hepatitis C virus RNA helicase. Journal of Virology 71, 9400-9409.
    [Google Scholar]
  22. Kim, J. R., Morgernstern, K. A., Griffith, J. P., Dwyer, M. D., Thomson, J. A., Murcko, M. A., Lin, C. & Caron, P. R. ( 1998; ). Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure 6, 89-100.[CrossRef]
    [Google Scholar]
  23. Kolykhalov, A. A., Feinstone, S. M. & Rice, C. M. ( 1996; ). Identification of a highly conserved sequence element at the 3′ terminus of hepatitis C virus genome RNA. Journal of Virology 70, 3363-3371.
    [Google Scholar]
  24. Korangy, F. & Julin, D. A. ( 1992; ). A mutation in the consensus ATP-binding sequence of the RecD subunit reduces the processivity of the RecBCD enzyme from Escherichia coli. Journal of Biological Chemistry 267, 3088-3095.
    [Google Scholar]
  25. Korangy, F. & Julin, D. A. ( 1993; ). Kinetics and processivity of ATP hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli. Biochemistry 32, 4873-4880.[CrossRef]
    [Google Scholar]
  26. Korolev, S., Hsieh, J., Gauss, G. H., Lohman, T. M. & Waksman, G. ( 1997; ). Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP. Cell 90, 635-647.[CrossRef]
    [Google Scholar]
  27. Korolev, S., Yao, N., Lohman, T. M., Weber, P. C. & Waksman, G. ( 1998; ). Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super-families of helicases. Protein Science 7, 605-610.[CrossRef]
    [Google Scholar]
  28. Kwong, A. D., Kim, J. L., Rao, G., Lipovsek, D. & Raybuck, S. A. ( 1998; ). Hepatitis C virus NS3/4A protease. Antiviral Research 40, 1-18.[CrossRef]
    [Google Scholar]
  29. Lohman, T. M. & Bjornson, K. P. ( 1996; ). Mechanisms of helicase-catalyzed DNA unwinding. Annual Review of Biochemistry 65, 169-214.[CrossRef]
    [Google Scholar]
  30. Lohmann, V., Koch, J. O. & Bartenschlager, R. ( 1996; ). Processing pathways of the hepatitis C virus proteins. Journal of Hepatology 24, 11-19.
    [Google Scholar]
  31. Lohmann, V., Körner, F., Herian, U. & Bartenschlager, R. ( 1997; ). Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. Journal of Virology 71, 8416-8428.
    [Google Scholar]
  32. Oh, J.-W., Ito, T. & Lai, M. M. C. ( 1999; ). A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA. Journal of Virology 73, 7694-7702.
    [Google Scholar]
  33. Phillips, R. J., Hickleton, D. C., Boehmer, P. E. & Emmerson, P. T. ( 1997; ). The RecB protein of Escherichia coli translocates along single-stranded DNA in the 3′ to 5′ direction: a proposed ratchet mechanism. Molecular and General Genetics 254, 319-329.
    [Google Scholar]
  34. Porter, D. J. T. ( 1998; ). A kinetic analysis of the oligonucleotide-modulated ATPase activity of the helicase domain of the NS3 protein from hepatitis C virus. Journal of Biological Chemistry 273, 14247-14253.[CrossRef]
    [Google Scholar]
  35. Porter, D. J. T., Short, S. A., Hanlon, M. H., Preugschat, F., Wilson, J. E., Willard, D. H.Jr & Consler, T. G. ( 1998; ). Product release is the major contributor to k cat for the hepatitis C virus helicase-catalyzed strand separation of short duplex DNA. Journal of Biological Chemistry 273, 18906-18914.[CrossRef]
    [Google Scholar]
  36. Preugschat, F., Averett, D. R., Clarke, B. E. & Porter, D. J. T. ( 1996; ). A steady-state and pre-steady state kinetic analysis of the NTPase activity associated with the hepatitis C virus NS3 helicase domain. Journal of Biological Chemistry 271, 24449-24457.[CrossRef]
    [Google Scholar]
  37. Rice, C. M. ( 1996; ). Flaviviridae: the viruses and their replication. In Fields Virology, pp. 931-960. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. New York: Raven Press.
  38. Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. ( 1998; ). Use of the T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology 185, 60-89.
    [Google Scholar]
  39. Subramanya, H. S., Bird, L. E., Brannigan, J. A. & Wigley, D. B. ( 1996; ). Crystal structure of a DExx box helicase. Nature 384, 379-383.[CrossRef]
    [Google Scholar]
  40. Tai, C.-L., Chi, W.-K., Chen, D.-S. & Hwang, L.-H. ( 1996; ). The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3). Journal of Virology 70, 8477-8484.
    [Google Scholar]
  41. Tanaka, T., Kato, N., Cho, M. J. & Shimotohno, K. ( 1996; ). Structure of the 3′ terminus of the hepatitis C virus. Journal of Virology 70, 3307-3312.
    [Google Scholar]
  42. Tsuchihara, K., Tanaka, T., Hijikata, M., Kuge, S., Toyoda, H., Nomoto, A., Yamamoto, N. & Shimotohno, K. ( 1997; ). Specific interaction of polypyrimidine tract-binding protein with the extreme 3′-terminal structure of the hepatitis C virus genome, the 3′X. Journal of Virology 71, 6720-6726.
    [Google Scholar]
  43. Velankar, S. S., Soultanas, P., Dillingham, M. S., Subramanya, H. S. & Wigley, D. B. ( 1999; ). Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell 97, 75-84.[CrossRef]
    [Google Scholar]
  44. 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. Journal of General Virology 80, 701-709.
    [Google Scholar]
  45. Wong, I. & Lohman, T. M. ( 1992; ). Allosteric effects of nucleotide cofactors on Escherichia coli Rep helicase-DNA binding. Science 256, 350-355.[CrossRef]
    [Google Scholar]
  46. Yao, N., Hesson, T., Cable, M., Hong, Z., Kwong, A. D., Le, H. V. & Weber, P. C. ( 1997; ). Structure of the hepatitis C virus RNA helicase domain. Nature Structural Biology 4, 463-467.[CrossRef]
    [Google Scholar]
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