RNA-dependent RNA polymerase activity encoded by GB virus-B non-structuralprotein 5B

Phylogenetic analysis and polyprotein organization comparison have shown th at GB virus-B (GBV-B) is closely related to hepatitis C virus (HCV). In thi s study, the coding region for GBV-B non-structural protein 5B (NS5B) was i solated by reverse transcription-polymerase chain reaction (RT-PCR) from po oled serum of GBV-B-infected tamarins. Expression of soluble GBV-B NS5B pro tein in Escherichia coli was achieved by removal of a 19-amino acid hydroph obic domain at the C-terminus of the protein. The truncated GBV-B NS5B (NS5 B Delta CT19) was purified to homogeneity and shown to possess an RNA-depen dent RNA polymerase (RdRp) activity in both gel-based and scintillation pro ximity assays. NS5B Delta CT19 required the divalent cation Mn2+ for enzyma tic activity, at an optimal concentration of 15 mM. Interestingly, Mg2+, at concentrations up to 20 mM, did not support the GBV-B NS5B activity. This differs from HCV NS5B where both Mn2+ and Mg2+ can support RdRp activity. Z n2+ was found to inhibit the activity of GBV-B NS5B, with a 50% inhibitory concentration (IC50) of 5-10 mu M. Higher concentrations of monovalent salt s (NaCl or KCl > 100 mM) and glycerol (> 3%) were also inhibitory. NS5B Del ta CT19 was able to bind to RNA homopolymers, but utilized most efficiently poly(C), the one with the lowest binding affinity for RNA synthesis. Mutat ional analysis of GBV-B NS5B demonstrated the importance of several conserv ed sequence motifs for enzymatic activity. Based on sequence homology (appr oximate to 37% identity and 52% similarity) between GBV-B and HCV NS5B prot eins, the active GBV-B RdRp provides a good surrogate assay system for HCV polymerase studies.