Nonstructural protein 3 (NS3) of hepatitis C virus contains a bipartite structure consisting of an N-terminal serine protease and a C-terminal DEXH box helicase. To investigate the roles of individual amino acid residues in the overall mechanism of unwinding, a mutational–functional analysis was performed based on a molecular model of the NS3 helicase domain bound to ssDNA, which has largely been confirmed by a recently published crystal structure of the NS3 helicase–ssDNA complex. Three full-length mutated NS3 proteins containing Tyr(392)Ala, Val(432)Gly and Trp(501)Ala single substitutions, respectively, together with a Tyr(392)Ala/Trp(501)Ala double-substituted protein were expressed in and purified to homogeneity. All individually mutated forms showed a reduction in duplex unwinding activity, single-stranded polynucleotide binding capacity and polynucleotide-stimulated ATPase activity compared to wild-type, though to different extents. Simultaneous replacement of both Tyr and Trp with Ala completely abolished all these enzymatic functions. On the other hand, the introduced amino acid substitutions had no influence on NS3 intrinsic ATPase activity and proteolytic efficiency. The results obtained with Trp(501)Ala and Val(432)Gly single-substituted enzymes are in agreement with a recently proposed model for NS3 unwinding activity. The mutant phenotype of the Tyr(392)Ala and Tyr(392)Ala/Trp(501)Ala enzymes, however, represents a completely novel finding.


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  1. Alter, M. J. (1997). Epidemiology of hepatitis C. Hepatology 26, 62S-65S.[CrossRef] [Google Scholar]
  2. 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]
  3. 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]
  4. Choo, Q.-L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W. & Houghton, M. (1989). Isolation of a cDNA clone derived from a blood-borne non-A non-B viral hepatitis genome. Science 244, 359-362.[CrossRef] [Google Scholar]
  5. Clarke, B. (1997). Molecular virology of hepatitis C virus. Journal of General Virology 78, 2397-2410. [Google Scholar]
  6. 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.
  7. 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]
  8. 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]
  9. 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]
  10. Gorbalenya, A. E., Koonin, E. V., Donchenko, A. P. & Blinov, V. M. (1989). Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Research 17, 4713-4730.[CrossRef] [Google Scholar]
  11. Gorbalenya, A. E., Koonin, E. V. & Wolf, Y. I. (1990). A new family of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses. FEBS Letters 262, 145-148.[CrossRef] [Google Scholar]
  12. 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]
  13. 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]
  14. Heilek, G. M. & Peterson, M. G. (1997). A point mutation abolishes the helicase but not the nucleoside triphosphatase activity of hepatitis C virus NS3 protein. Journal of Virology 71, 6264-6266. [Google Scholar]
  15. Jin, L. & Peterson, D. L. (1995). Expression, isolation, and characterization of the hepatitis C virus ATPase/RNA helicase. Archives of Biochemistry and Biophysics 323, 47-53.[CrossRef] [Google Scholar]
  16. Kadaré, G. & Haenni, A.-L. (1997). Virus-encoded RNA helicases.Journal of Virology 71, 2583-2590. [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. Korolev, S., Hsieh, J., Gauss, G. H., Lohman, T. M. & Waksman, G. (1997). Major domain swivelling 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]
  22. 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]
  23. 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]
  24. Lin, C. & Kim, J. L. (1999). Structure-based mutagenesis study of hepatitis C virus NS3 helicase. Journal of Virology 73, 8798-8807. [Google Scholar]
  25. Lohman, T. M. & Bjornson, K. P. (1996). Mechanisms of helicase-catalyzed DNA unwinding. Annual Review of Biochemistry 65, 169-214.[CrossRef] [Google Scholar]
  26. Lohmann, V., Koch, J. O. & Bartenschlager, R. (1996). Processing pathways of the hepatitis C virus proteins. Journal of Hepatology 24, 11-19. [Google Scholar]
  27. Lüking, A., Stahl, U. & Schmidt, U. (1998). The protein family of RNA helicases. Critical Reviews in Biochemistry and Molecular Biology 33, 259-296.[CrossRef] [Google Scholar]
  28. Matson, S. W., Bean, D. W. & George, J. W. (1994). DNA helicases: enzymes with essential roles in all aspects of DNA metabolism. Bioassays 16, 13-22.[CrossRef] [Google Scholar]
  29. Paolini, C., De Francesco, R. & Gallinari, P. (2000). Enzymatic properties of hepatitis C virus NS3-associated helicase. Journal of General Virology 81, 1335-1345. [Google Scholar]
  30. 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]
  31. 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 kcat for the hepatitis C virus helicase-catalyzed strand separation of short duplex DNA.Journal of Biological Chemistry 273, 18906-18914.[CrossRef] [Google Scholar]
  32. 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]
  33. 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. Philadelphia: Lippincott–Raven.
  34. 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]
  35. 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]
  36. Suzich, J. A., Tamura, J. K., Palmer Hill, F., Warrener, P., Grakoui, A., Rice, C. M., Feinstone, S. M. & Collett, M. S. (1993). Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes. Journal of Virology 67, 6152-6158. [Google Scholar]
  37. 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]
  38. 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]
  39. 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]
  40. 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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