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Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli.
- Source :
-
Journal of virology [J Virol] 1999 Feb; Vol. 73 (2), pp. 1649-54. - Publication Year :
- 1999
-
Abstract
- Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BDeltaCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.
- Subjects :
- Amino Acid Sequence
Cations, Divalent
Enzyme Inhibitors pharmacology
Escherichia coli
Gliotoxin pharmacology
Humans
Metals
Molecular Sequence Data
RNA-Dependent RNA Polymerase antagonists & inhibitors
RNA-Dependent RNA Polymerase genetics
RNA-Dependent RNA Polymerase isolation & purification
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins isolation & purification
Recombinant Fusion Proteins metabolism
Sequence Deletion
Solubility
Viral Nonstructural Proteins antagonists & inhibitors
Viral Nonstructural Proteins genetics
Viral Nonstructural Proteins isolation & purification
Hepacivirus enzymology
RNA-Dependent RNA Polymerase metabolism
Viral Nonstructural Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0022-538X
- Volume :
- 73
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Journal of virology
- Publication Type :
- Academic Journal
- Accession number :
- 9882374
- Full Text :
- https://doi.org/10.1128/JVI.73.2.1649-1654.1999