EXPRESSION OF RECOMBINANT HEPATITIS-C VIRUS NONSTRUCTURAL PROTEIN 5B IN ESCHERICHIA-COLI

The hepatitis C virus (HCV) represents a major public health problem t hat can produce liver failure and hepatocellular carcinoma in chronica lly infected patients. Our goal was to express the HCV non-structural protein 5B (NS5B) protein of HCV genotype 1a in Escherichia coli and i nitiate studies of its role in HCV genomic replication. In this report we demonstrate that a recombinant NS5B protein with an amino terminal sequence of ASMSYSWTG has RNA-dependent RNA polymerase (RDRP) activit y. This recombinant enzyme was active in poly(U) polymerase assays and produced template-sized RNA products when globin mRNA was used as a t emplate. The polymerase activity of recombinant NS5B was primer-depend ent and was active for at least 60 min of incubation at 30 degrees C. Deletion of the carboxyl terminal region of HCV NS5B resulted in a los s of RDRP activity indicating that the enzymatic activity observed was due to the full-length recombinant enzyme. Recombinant NS5B (RDRP) sh ould assist in understanding the mechanism of HCV replication and the identification of specific enzyme inhibitors. (C) 1998 Elsevier Scienc e B.V. All rights reserved.