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

Fingerprint

RNA Helicases
Hepacivirus
Adenosine Triphosphate
RNA
Recombinant Proteins
Adenosine Triphosphatases
Proteins
Polynucleotides
Conserved Sequence
Nickel
Histidine
Alanine
Sepharose
Arginine
Chromatography
Hydrolysis
Escherichia coli
Mutation
Enzymes
Genes

Keywords

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

ASJC Scopus subject areas

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

Cite this

Functional interactions between conserved motifs of the hepatitis C virus RNA helicase protein NS3. / Min, Kyung Hyun; Sung, Young Chul; Choi, Sang-Yun; Ahn, Byung-Yoon.

In: Virus Genes, Vol. 19, No. 1, 09.09.1999, p. 33-43.

Research output: Contribution to journalArticle

@article{e7794c6dab8d4fd8b0bf08e88aa57784,
title = "Functional interactions between conserved motifs of the hepatitis C virus RNA helicase protein NS3",
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.",
keywords = "ATPase, Hepatitis C virus, Mutagenesis, NS3 gene, RNA helicase",
author = "Min, {Kyung Hyun} and Sung, {Young Chul} and Sang-Yun Choi and Byung-Yoon Ahn",
year = "1999",
month = "9",
day = "9",
doi = "10.1023/A:1008184522153",
language = "English",
volume = "19",
pages = "33--43",
journal = "Virus Genes",
issn = "0920-8569",
publisher = "Springer Netherlands",
number = "1",

}

TY - JOUR

T1 - Functional interactions between conserved motifs of the hepatitis C virus RNA helicase protein NS3

AU - Min, Kyung Hyun

AU - Sung, Young Chul

AU - Choi, Sang-Yun

AU - Ahn, Byung-Yoon

PY - 1999/9/9

Y1 - 1999/9/9

N2 - 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.

AB - 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.

KW - ATPase

KW - Hepatitis C virus

KW - Mutagenesis

KW - NS3 gene

KW - RNA helicase

UR - http://www.scopus.com/inward/record.url?scp=0032853885&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032853885&partnerID=8YFLogxK

U2 - 10.1023/A:1008184522153

DO - 10.1023/A:1008184522153

M3 - Article

C2 - 10499448

AN - SCOPUS:0032853885

VL - 19

SP - 33

EP - 43

JO - Virus Genes

JF - Virus Genes

SN - 0920-8569

IS - 1

ER -