Characterization of soluble hepatitis C virus RNA-dependent RNA polymeraseexpressed in Escherichia coli

Production of soluble full-length nonstructural protein 5B (NS5B) of hepati tis C virus (HCV) has been shown to be problematic and requires the additio n of salts, glycerol, and detergents. In an effort to improve the solubilit y of NS5B, the hydrophobic C terminus containing 21 amino acids was removed , yielding a truncated NS5B (NS5B Delta CT) which is highly soluble and mon odispersed in the absence of detergents. Fine deletional analysis of this r egion revealed that a four-leucine motif (LLLL) in the hydrophobic domain i s responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) act ivity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are pr eferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibi ted HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed tha t HCV NS5B has a rather low processivity compared to those of other known p olymerases.