Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Sep;75(17):8289-97.
doi: 10.1128/jvi.75.17.8289-8297.2001.

Structure-based mutational analysis of the hepatitis C virus NS3 helicase

C L Tai  1 , W C Pan, S H Liaw, U C Yang, L H Hwang, D S Chen
Affiliations

Structure-based mutational analysis of the hepatitis C virus NS3 helicase

C L Tai et al. J Virol. 2001 Sep.

Abstract

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.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Site-directed mutagenesis of the HCV NS3 helicase domain. (A) Summary of all mutant clones. The top line sequences represent conserved motifs I, II, III, and VI, respectively, of HCV NS3 helicase. The amino acid residue replacements are shown below the arrows. (B) Purities of mutant proteins. The wild-type and mutant proteins were expressed in E. coli and purified as described in Materials and Methods. Two micrograms of each of the purified proteins was resolved by SDS–12.5% PAGE and stained with Coomassie brilliant blue. WT denotes the wild-type helicase protein.
FIG. 2
FIG. 2
ATP binding activity of the wild type and some mutant helicase proteins. One microgram of the wild-type or mutant helicase proteins was incubated with [α-32P]ATP in the presence of poly(U) and was UV cross-linked [WT(-UV)] as described in Materials and Methods. Samples were separated by SDS–12.5% PAGE. Following electrophoresis, the gels were stained with Coomassie brilliant blue, dried, and processed for autoradiography. WT, wild-type helicase protein; BSA, bovine serum albumin.
FIG. 3
FIG. 3
RNA binding activity of wild-type and mutant helicase proteins. The wild-type or mutant helicase proteins, cold release strand RNA, and labeled dsRNA substrate were incubated as described in Materials and Methods. The reaction mixtures were electrophoresed by native PAGE and were processed for autoradiography. WT denotes the wild-type helicase protein.
FIG. 4
FIG. 4
Relationship of ATPase activity and RNA helicase activity. Both ATPase activity and RNA helicase activity of all mutant proteins were translated into ratios relative to activity of the wild-type (WT) helicase protein (data shown in Table 2). The relationship was then plotted as the RNA helicase activity versus ATPase activity.
FIG. 5
FIG. 5
Locations of the important amino acid residues in the three-dimensional conformation. The structure of HCV NS3 helicase was based on that determined by Kim et al. (22) using Protein Data Bank (PDB) accession code 1A1V. The important residues identified herein and the Arg-464/Arg-467 previously demonstrated (21, 22, 43) are emphasized by being represented in stick format, while the sulfate ion is represented in Corey-Pauling-Koltun (CPK) format. Residues His-293 and Gln-460 are located in domains I and II, respectively. It is hypothesized that the amine group of the histidine ring of His-293 formed a hydrogen bond with the carbonyl oxygen of the side chain of Gln-460. Residue Arg-461 is located in the interior of domain II. The inset indicates that the Arg-461 can form hydrogen bonds with Asp-412 and Asp-427, which are located at conserved motif V. These figures were drawn using InsightII.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Black M E, Hruby D E. Site-directed mutagenesis of a conserved domain in vaccinia virus thymidine kinase. Evidence for a potential role in magnesium binding. J Biol Chem. 1992;267:6801–6806. - PubMed
    1. Brillanti S, Garson J, Foli M, Whitby K, Deaville R, Masci C, Miglioli M, Barbara L. A pilot study of combination therapy with ribavirin plus interferon alfa for interferon alfa-resistant chronic hepatitis C. Gastroenterology. 1994;107:812–817. - PubMed
    1. Brillanti S, Miglioli M, Barbara L. Combination antiviral therapy with ribavirin and interferon alfa in interferon alfa relapsers and non-responders: Italian experience. J Hepatol. 1995;23:13–15. - PubMed
    1. Chamberlain R W, Adams N, Saeed A A, Simmonds P, Elliott R M. Complete nucleotide sequence of a type 4 hepatitis C virus variant, the predominant genotype in the Middle East. J Gen Virol. 1997;78:1341–1347. - PubMed
    1. Chemello L, Cavalletto L, Bernardinello E, Guido M, Pontisso P, Alberti A. The effect of interferon alfa and ribavirin combination therapy in naive patients with chronic hepatitis C. J Hepatol. 1995;23:8–12. - PubMed

Publication types

MeSH terms

LinkOut - more resources