title: Enzymatic activities of the GB virus-B RNA-dependent RNA polymerase.
abstract: The GB virus-B (GBV-B) nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) with greater than 50% sequence similarity to the hepatitis C virus (HCV) NS5B. Recombinant GBV-B NS5B was reported to possess RdRp activity (W. Zhong et al., 2000, J. Viral Hepat. 7, 335-342). In this study, the GBV-B RdRp was examined more thoroughly for different RNA synthesis activities, including primer-extension, de novo initiation, template switch, terminal nucleotide addition, and template specificity. The results can be compared with previous characterizations of the HCV RdRp. The two RdRps share similarities in terms of metal ion and template preference, the abilities to add nontemplated nucleotides, perform both de novo initiation and extension from a primer, and switch templates. However, several differences in RNA synthesis between the GBV-B and HCV RdRps were observed, including (i) optimal temperatures for activity, (ii) ranges of Mn(2+) concentration tolerated for activity, and (iii) cation requirements for de novo RNA synthesis and terminal transferase activity. To assess whether the recombinant GBV-B RdRp may represent a relevant surrogate system for testing HCV antiviral agents, two compounds demonstrated to be active at nanomolar concentrations against HCV NS5B were tested on the GBV RdRp. A chain terminating nucleotide analog could prevent RNA synthesis, while a nonnucleoside HCV inhibitor was unable to affect RNA synthesis by the GBV RdRp.
authors.affiliation: Department of Biology, Indiana University, Bloomington, IN 47405, USA.
authors.forename: C T
authors.initials: CT
authors.lastname: Ranjith-Kumar
authors.forename: Jan Lee
authors.initials: JL
authors.lastname: Santos
authors.forename: Lester L
authors.initials: LL
authors.lastname: Gutshall
authors.forename: Victor K
authors.initials: VK
authors.lastname: Johnston
authors.forename: Juili
authors.initials: J
authors.lastname: Lin-Goerke
authors.forename: Min-Ju
authors.initials: MJ
authors.lastname: Kim
authors.forename: David J
authors.initials: DJ
authors.lastname: Porter
authors.forename: Derrick
authors.initials: D
authors.lastname: Maley
authors.forename: Cathy
authors.initials: C
authors.lastname: Greenwood
authors.forename: David L
authors.initials: DL
authors.lastname: Earnshaw
authors.forename: Audrey
authors.initials: A
authors.lastname: Baker
authors.forename: Baohua
authors.initials: B
authors.lastname: Gu
authors.forename: Carol
authors.initials: C
authors.lastname: Silverman
authors.forename: Robert T
authors.initials: RT
authors.lastname: Sarisky
authors.forename: Cheng
authors.initials: C
authors.lastname: Kao
mesh_terms: D001483:Base Sequence; D029406:GB virus B / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013698:Templates, Genetic
chemical_list: D009711:Nucleotides; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase