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Journal of Virology, September 2001, p. 8289-8297, Vol. 75, No. 17
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.17.8289-8297.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

Chun-Ling Tai,1 Wen-Ching Pan,1 Shwu-Huey Liaw,2 Ueng-Cheng Yang,3 Lih-Hwa Hwang,1,4,* and Ding-Shinn Chen4

Graduate Institute of Microbiology, National Taiwan University,1 Department of Life Science2 and Institute of Biochemistry,3 National Yang Ming University, and Hepatitis Research Center, National Taiwan University Hospital,4 Taipei, Taiwan

Received 2 January 2001/Accepted 11 May 2001

The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3.

* Corresponding author. Mailing address: Hepatitis Research Center, National Taiwan University Hospital, 7, Chung-Shan S. Rd., Taipei 100, Taiwan. Phone: 886-2-23123456, ext. 7503. Fax: 886-2-23825962. E-mail: lihhwa{at}ha.mc.ntu.edu.tw.

Journal of Virology, September 2001, p. 8289-8297, Vol. 75, No. 17
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.17.8289-8297.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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