Functional interactions between conserved motifs of the hepatitis C virus RNA helicase protein NS3

Kyung Hyun Min, Young Chul Sung, Sang-Yun Choi, Byung-Yoon Ahn

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

The hepatitis C virus NS3 gene encodes a RNA helicase with several sequence motifs conserved among the members of the DExH box protein family. The contributions of the sequence motifs to enzyme activity were assessed in this study by substitution of alanine for the Lys in the ATP binding motif GxGK (referred to as K1236A mutation), or for the Asp in the DExH motif (D1316A), or for the Arg in the middle of the QRxGRxGR motif known for RNA binding (R1490A). Histidine-tagged recombinant proteins of Mr 54,000 were expressed in Escherichia coli and purified by chromatography on nickel agarose. All three mutants were severely defective in ATPase and RNA helicase activities, but loss of the ATPase activity was not dependent on polynucleotide cofactors. With the exception of R1490A mutant, a stable complex was formed between dsRNA substrates and recombinant proteins, indicating that the arginine-rich motif is required for efficient RNA binding. Complex formation was not affected by omission of ATP or substitution by a non-hydrolyzable analog AMP-PCP, suggesting that neither binding nor hydrolysis of ATP is required for RNA binding. Moreover, the K1236A mutant which was defective in binding ATP exhibited an unusually strong affinity for RNA duplex. These results suggest that the conserved motifs cooperatively constitute a large functional domain rather than act as individual domains with strictly independent functions, and that alteration of one motif affects functions of other motifs in a mutually interactive fashion.

Original languageEnglish
Pages (from-to)33-43
Number of pages11
JournalVirus Genes
Volume19
Issue number1
DOIs
Publication statusPublished - 1999 Sep 9

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Keywords

  • ATPase
  • Hepatitis C virus
  • Mutagenesis
  • NS3 gene
  • RNA helicase

ASJC Scopus subject areas

  • Virology
  • Molecular Biology
  • Immunology
  • Applied Microbiology and Biotechnology
  • Genetics

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