This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lin, C.
Right arrow Articles by Kim, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lin, C.
Right arrow Articles by Kim, J. L.

 Previous Article  |  Next Article 

Journal of Virology, October 1999, p. 8798-8807, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Structure-Based Mutagenesis Study of Hepatitis C Virus NS3 Helicase

Chao Lin* and Joseph L. Kim

Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139

Received 14 April 1999/Accepted 30 June 1999

The NS3 protein of hepatitis C virus (HCV) is a bifunctional protein containing a serine protease in the N-terminal one-third, which is stimulated upon binding of the NS4A cofactor, and an RNA helicase in the C-terminal two-thirds. In this study, a C-terminal hexahistidine-tagged helicase domain of the HCV NS3 protein was expressed in Escherichia coli and purified to homogeneity by conventional chromatography. The purified HCV helicase domain has a basal ATPase activity, a polynucleotide-stimulated ATPase activity, and a nucleic acid unwinding activity and binds efficiently to single-stranded polynucleotide. Detailed characterization of the purified HCV helicase domain with regard to all four activities is presented. Recently, we published an X-ray crystallographic structure of a binary complex of the HCV helicase with a (dU)8 oligonucleotide, in which several conserved residues of the HCV helicase were shown to be involved in interactions between the HCV helicase and oligonucleotide. Here, site-directed mutagenesis was used to elucidate the roles of these residues in helicase function. Four individual mutations, Thr to Ala at position 269, Thr to Ala at position 411, Trp to Leu at position 501, and Trp to Ala at position 501, produced a severe reduction of RNA binding and completely abolished unwinding activity and stimulation of ATPase activity by poly(U), although the basal ATPase activity (activity in the absence of polynucleotide) of these mutants remained intact. Alanine substitution at Ser-231 or Ser-370 resulted in enzymes that were indistinguishable from wild-type HCV helicase with regard to all four activities. A mutant bearing Phe at Trp-501 showed wild-type levels of basal ATPase, unwinding activity, and single-stranded RNA binding activity. Interestingly, ATPase activity of this mutant became less responsive to stimulation by poly(U) but not to stimulation by other polynucleotides, such as poly(C). Given the conservation of some of these residues in other DNA and RNA helicases, their role in the mechanism of unwinding of double-stranded nucleic acid is discussed.

* Corresponding author. Mailing address: Vertex Pharmaceuticals Incorporated, 130 Waverly St., Cambridge, MA 02139. Phone: (617) 577-6000. Fax: (617) 577-6210. E-mail: Lin{at}vpharm.com.

Journal of Virology, October 1999, p. 8798-8807, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Bamming, D., Horvath, C. M. (2009). Regulation of Signal Transduction by Enzymatically Inactive Antiviral RNA Helicase Proteins MDA5, RIG-I, and LGP2. J. Biol. Chem. 284: 9700-9712 [Abstract] [Full Text]  
  • Roy, P. (2008). Bluetongue virus: dissection of the polymerase complex. J. Gen. Virol. 89: 1789-1804 [Abstract] [Full Text]  
  • Ma, Y., Yates, J., Liang, Y., Lemon, S. M., Yi, M. (2008). NS3 Helicase Domains Involved in Infectious Intracellular Hepatitis C Virus Particle Assembly. J. Virol. 82: 7624-7639 [Abstract] [Full Text]  
  • Myong, S., Bruno, M. M., Pyle, A. M., Ha, T. (2007). Spring-Loaded Mechanism of DNA Unwinding by Hepatitis C Virus NS3 Helicase. Science 317: 513-516 [Abstract] [Full Text]  
  • Sampath, A., Xu, T., Chao, A., Luo, D., Lescar, J., Vasudevan, S. G. (2006). Structure-based mutational analysis of the NS3 helicase from dengue virus.. J. Virol. 80: 6686-6690 [Abstract] [Full Text]  
  • Lam, A. M. I., Frick, D. N. (2006). Hepatitis C Virus Subgenomic Replicon Requires an Active NS3 RNA Helicase. J. Virol. 80: 404-411 [Abstract] [Full Text]  
  • Wang, X., Lee, W.-M., Watanabe, T., Schwartz, M., Janda, M., Ahlquist, P. (2005). Brome Mosaic Virus 1a Nucleoside Triphosphatase/Helicase Domain Plays Crucial Roles in Recruiting RNA Replication Templates. J. Virol. 79: 13747-13758 [Abstract] [Full Text]  
  • Frick, D. N., Rypma, R. S., Lam, A. M. I., Frenz, C. M. (2004). Electrostatic analysis of the hepatitis C virus NS3 helicase reveals both active and allosteric site locations. Nucleic Acids Res 32: 5519-5528 [Abstract] [Full Text]  
  • Kumar, P., Sulochana, P., Nirmala, G., Haridattatreya, M., Satchidanandam, V. (2004). Conserved amino acids 193-324 of non-structural protein 3 are a dominant source of peptide determinants for CD4+ and CD8+ T cells in a healthy Japanese encephalitis virus-endemic cohort. J. Gen. Virol. 85: 1131-1143 [Abstract] [Full Text]  
  • Schneider, S., Campodonico, E., Schwer, B. (2004). Motifs IV and V in the DEAH Box Splicing Factor Prp22 Are Important for RNA Unwinding, and Helicase-defective Prp22 Mutants Are Suppressed by Prp8. J. Biol. Chem. 279: 8617-8626 [Abstract] [Full Text]  
  • Frick, D. N., Rypma, R. S., Lam, A. M. I., Gu, B. (2004). The Nonstructural Protein 3 Protease/Helicase Requires an Intact Protease Domain to Unwind Duplex RNA Efficiently. J. Biol. Chem. 279: 1269-1280 [Abstract] [Full Text]  
  • Worthey, E. A., Schnaufer, A., Mian, I. S., Stuart, K., Salavati, R. (2003). Comparative analysis of editosome proteins in trypanosomatids. Nucleic Acids Res 31: 6392-6408 [Abstract] [Full Text]  
  • Lam, A. M. I., Keeney, D., Frick, D. N. (2003). Two Novel Conserved Motifs in the Hepatitis C Virus NS3 Protein Critical for Helicase Action. J. Biol. Chem. 278: 44514-44524 [Abstract] [Full Text]  
  • Kar, A. K., Roy, P. (2003). Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6. J. Virol. 77: 11347-11356 [Abstract] [Full Text]  
  • OGURO, A., OHTSU, T., SVITKIN, Y. V., SONENBERG, N., NAKAMURA, Y. (2003). RNA aptamers to initiation factor 4A helicase hinder cap-dependent translation by blocking ATP hydrolysis. RNA 9: 394-407 [Abstract] [Full Text]  
  • Lam, A. M. I., Keeney, D., Eckert, P. Q., Frick, D. N. (2003). Hepatitis C Virus NS3 ATPases/Helicases from Different Genotypes Exhibit Variations in Enzymatic Properties. J. Virol. 77: 3950-3961 [Abstract] [Full Text]  
  • Marintcheva, B., Weller, S. K. (2003). Helicase Motif Ia Is Involved in Single-Strand DNA-Binding and Helicase Activities of the Herpes Simplex Virus Type 1 Origin-Binding Protein, UL9. J. Virol. 77: 2477-2488 [Abstract] [Full Text]  
  • Kim, J. W., Seo, M. Y., Shelat, A., Kim, C. S., Kwon, T. W., Lu, H.-h., Moustakas, D. T., Sun, J., Han, J. H. (2002). Structurally Conserved Amino Acid W501 Is Required for RNA Helicase Activity but Is Not Essential for DNA Helicase Activity of Hepatitis C Virus NS3 Protein. J. Virol. 77: 571-582 [Abstract] [Full Text]  
  • Matusan, A. E., Pryor, M. J., Davidson, A. D., Wright, P. J. (2001). Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication. J. Virol. 75: 9633-9643 [Abstract] [Full Text]  
  • Rho, J., Choi, S., Seong, Y. R., Choi, J., Im, D.-S. (2001). The Arginine-1493 Residue in QRRGRTGR1493G Motif IV of the Hepatitis C Virus NS3 Helicase Domain Is Essential for NS3 Protein Methylation by the Protein Arginine Methyltransferase 1. J. Virol. 75: 8031-8044 [Abstract] [Full Text]  
  • Tai, C.-L., Pan, W.-C., Liaw, S.-H., Yang, U.-C., Hwang, L.-H., Chen, D.-S. (2001). Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase. J. Virol. 75: 8289-8297 [Abstract] [Full Text]  
  • Dillingham, M. S., Soultanas, P., Wiley, P., Webb, M. R., Wigley, D. B. (2001). Defining the roles of individual residues in the single-stranded DNA binding site of PcrA helicase. Proc. Natl. Acad. Sci. USA 98: 8381-8387 [Abstract] [Full Text]  
  • Borowski, P., Niebuhr, A., Mueller, O., Bretner, M., Felczak, K., Kulikowski, T., Schmitz, H. (2001). Purification and Characterization of West Nile Virus Nucleoside Triphosphatase (NTPase)/Helicase: Evidence for Dissociation of the NTPase and Helicase Activities of the Enzyme. J. Virol. 75: 3220-3229 [Abstract] [Full Text]  
  • Banerjee, R., Dasgupta, A. (2001). Specific Interaction of Hepatitis C Virus Protease/Helicase NS3 with the 3'-Terminal Sequences of Viral Positive- and Negative-Strand RNA. J. Virol. 75: 1708-1721 [Abstract] [Full Text]  
  • Khu, Y.-L., Koh, E., Lim, S. P., Tan, Y. H., Brenner, S., Lim, S. G., Hong, W. J., Goh, P.-Y. (2001). Mutations That Affect Dimer Formation and Helicase Activity of the Hepatitis C Virus Helicase. J. Virol. 75: 205-214 [Abstract] [Full Text]  
  • Chang, S. C., Cheng, J.-C., Kou, Y.-H., Kao, C.-H., Chiu, C.-H., Wu, H.-Y., Chang, M.-F. (2000). Roles of the AX4GKS and Arginine-Rich Motifs of Hepatitis C Virus RNA Helicase in ATP- and Viral RNA-Binding Activity. J. Virol. 74: 9732-9737 [Abstract] [Full Text]  
  • Paolini, C., Lahm, A., De Francesco, R., Gallinari, P. (2000). Mutational analysis of hepatitis C virus NS3-associated helicase. J. Gen. Virol. 81: 1649-1658 [Abstract] [Full Text]  
  • Rogers, G. W. Jr., Lima, W. F., Merrick, W. C. (2001). Further Characterization of the Helicase Activity of eIF4A. SUBSTRATE SPECIFICITY. J. Biol. Chem. 276: 12598-12608 [Abstract] [Full Text]  
  • Marintcheva, B., Weller, S. K. (2001). Residues within the Conserved Helicase Motifs of UL9, the Origin-binding Protein of Herpes Simplex Virus-1, Are Essential for Helicase Activity but Not for Dimerization or Origin Binding Activity. J. Biol. Chem. 276: 6605-6615 [Abstract] [Full Text]  
  • Rho, J., Choi, S., Seong, Y. R., Cho, W.-K., Kim, S. H., Im, D.-S. (2001). PRMT5, Which Forms Distinct Homo-oligomers, Is a Member of the Protein-arginine Methyltransferase Family. J. Biol. Chem. 276: 11393-11401 [Abstract] [Full Text]  
  • Oh, J.-W., Sheu, G.-T., Lai, M. M. C. (2000). Template Requirement and Initiation Site Selection by Hepatitis C Virus Polymerase on a Minimal Viral RNA Template. J. Biol. Chem. 275: 17710-17717 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»