Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli.
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.
Ferrari
E
E
Antiviral Therapy, Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539, USA.
Wright-Minogue
J
J
Fang
J W
JW
Baroudy
B M
BM
Lau
J Y
JY
Hong
Z
Z
D000595:Amino Acid Sequence; D002413:Cations, Divalent; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D005912:Gliotoxin / Q000494:pharmacology; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D008670:Metals; D008969:Molecular Sequence Data; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D012995:Solubility; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D008670:Metals; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D005912:Gliotoxin; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase