Abstract
The non-structural protein 3 (NS3) of hepatitis C virus (HCV) possesses three activities which are likely to be essential for virus replication; a serine protease located in the N terminus and helicase and NTPase activities located in the C terminus. Sequence analysis of the helicase/NTPase domain has identified motifs indicative of the DEAD-box family of helicases. Here we present the characterization of the helicase and NTPase activities of full-length NS3, expressed as a His-tagged fusion protein in E. coli, and make comparisons with published data of NS3 helicase domain alone. The helicase and NTPase activities of full-length NS3 have been demonstrated and we have characterized the effects of amino acid substitutions on conserved motifs of NS3 helicase. Helicase and NTPase activities were dependent on Mg2+ and ATP and inhibited by monovalent cations. NS3 was able to hydrolyse all four NTPs and dNTPs to drive DNA duplex unwinding but with differing abilities. NTPase activity was stimulated by all polynucleotides tested, with poly(U) having the greatest effect. Mutational analysis of conserved motifs of NS3 helicase showed all conserved residues to be required for optimal activity. These results are in accord with a recently proposed model for NS3 helicase activity.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Acid Anhydride Hydrolases / antagonists & inhibitors
-
Acid Anhydride Hydrolases / chemistry
-
Acid Anhydride Hydrolases / genetics
-
Acid Anhydride Hydrolases / metabolism*
-
Adenosine Triphosphate / analogs & derivatives
-
Adenosine Triphosphate / metabolism
-
Adenosine Triphosphate / pharmacology
-
Amino Acid Sequence
-
Amino Acid Substitution
-
Binding Sites
-
Cations / pharmacology
-
Conserved Sequence
-
DNA / metabolism
-
DNA Helicases / antagonists & inhibitors
-
DNA Helicases / chemistry
-
DNA Helicases / genetics
-
DNA Helicases / metabolism*
-
DNA Mutational Analysis
-
Escherichia coli / genetics
-
Hepacivirus / enzymology*
-
Hepacivirus / genetics
-
Hepatitis C / virology
-
Humans
-
Hydrogen-Ion Concentration
-
Kinetics
-
Magnesium / pharmacology
-
Nucleoside-Triphosphatase
-
Nucleotides / pharmacology
-
Recombinant Fusion Proteins / biosynthesis
-
Recombinant Fusion Proteins / isolation & purification
-
Recombinant Fusion Proteins / metabolism
-
Viral Nonstructural Proteins / antagonists & inhibitors
-
Viral Nonstructural Proteins / genetics
-
Viral Nonstructural Proteins / isolation & purification
-
Viral Nonstructural Proteins / metabolism*
Substances
-
Cations
-
NS3 protein, hepatitis C virus
-
Nucleotides
-
Recombinant Fusion Proteins
-
Viral Nonstructural Proteins
-
adenosine 5'-O-(3-thiotriphosphate)
-
Adenosine Triphosphate
-
DNA
-
Acid Anhydride Hydrolases
-
Nucleoside-Triphosphatase
-
DNA Helicases
-
Magnesium