Structure-based mutational analysis of the hepatitis C virus NS3 helicase
Journal
Journal of Virology
Journal Volume
75
Journal Issue
17
Pages
8289-8297
Date Issued
2001
Author(s)
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 Gin-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.
SDGs
Other Subjects
adenosine triphosphate; amino acid; helicase; mutant protein; article; carboxy terminal sequence; controlled study; enzyme activity; enzyme structure; Hepatitis C virus; hydrolysis; nonhuman; nucleotide sequence; point mutation; priority journal; protein expression; protein purification; RNA binding; virus mutation; Adenosine Triphosphatases; Hepacivirus; Models, Molecular; Point Mutation; Protein Conformation; RNA Helicases; RNA, Double-Stranded; RNA, Viral; Structure-Activity Relationship; Viral Nonstructural Proteins
Type
journal article