title: RNA-dependent RNA polymerase activity encoded by GB virus-B non-structural protein 5B.
abstract: Phylogenetic analysis and polyprotein organization comparison have shown that GB virus-B (GBV-B) is closely related to hepatitis C virus (HCV). In this study, the coding region for GBV-B non-structural protein 5B (NS5B) was isolated by reverse transcription-polymerase chain reaction (RT-PCR) from pooled serum of GBV-B-infected tamarins. Expression of soluble GBV-B NS5B protein in Escherichia coli was achieved by removal of a 19-amino acid hydrophobic domain at the C-terminus of the protein. The truncated GBV-B NS5B (NS5BDeltaCT19) was purified to homogeneity and shown to possess an RNA-dependent RNA polymerase (RdRp) activity in both gel-based and scintillation proximity assays. NS5BDeltaCT19 required the divalent cation Mn2+ for enzymatic 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. Zn2+ was found to inhibit the activity of GBV-B NS5B, with a 50% inhibitory concentration (IC50) of 5-10 microM. Higher concentrations of monovalent salts (NaCl or KCl > 100 mM) and glycerol (> 3%) were also inhibitory. NS5BDeltaCT19 was able to bind to RNA homopolymers, but utilized most efficiently poly(C), the one with the lowest binding affinity for RNA synthesis. Mutational analysis of GBV-B NS5B demonstrated the importance of several conserved sequence motifs for enzymatic activity. Based on sequence homology ( approximately 37% identity and 52% similarity) between GBV-B and HCV NS5B proteins, the active GBV-B RdRp provides a good surrogate assay system for HCV polymerase studies.
authors.affiliation: Department of Antiviral Therapy, Schering-Plough Research Institute, Kenilworth, NJ 07033-0539, USA.
authors.forename: W
authors.initials: W
authors.lastname: Zhong
authors.forename: P
authors.initials: P
authors.lastname: Ingravallo
authors.forename: J
authors.initials: J
authors.lastname: Wright-Minogue
authors.forename: A S
authors.initials: AS
authors.lastname: Uss
authors.forename: A
authors.initials: A
authors.lastname: Skelton
authors.forename: E
authors.initials: E
authors.lastname: Ferrari
authors.forename: J Y
authors.initials: JY
authors.lastname: Lau
authors.forename: Z
authors.initials: Z
authors.lastname: Hong
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004252:DNA Mutational Analysis; D018067:Flaviviridae / Q000201:enzymology* / Q000235:genetics; D006524:Hepatitis, Viral, Animal / Q000821:virology*; D008969:Molecular Sequence Data; D008992:Monkey Diseases / Q000821:virology; D012367:RNA, Viral / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012452:Saguinus; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D012367:RNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase