title: Phosphorylation at the N-terminal finger subdomain of a viral RNA-dependent RNA polymerase.
abstract: The RNA-dependent RNA polymerase (RdRP) of the Hepatitis C virus (HCV), named NS5B, is phosphorylated by the cellular protein kinase C-related kinase 2 (PRK2) at two serine residues (Ser29 and Ser42) of the finger subdomain (genotype 1b). Herein, using bioinformatics, we selected four potential phosphorylation residues (Ser46, Ser76, Ser96 and Ser112) of NS5B (genotype 2a) for study. Whereas the NS5B Ser46D and Ser76D substitutions seemed to improve polymerase activity, the Ser96D mutation decreased colony formation efficiency. Active WT NS5B was utilized in in vitro kinase assays, and phosphopeptides were analyzed by mass spectrometry. Interestingly, the data indicated that both the NS5B Ser29 and Ser76 residues resulted phosphorylated. Thus, as Ser76 is absolutely conserved across HCV genotypes, our results confirmed the relevance of these sites for both genotypes and suggested that Ser76 becomes phosphorylated by a cellular kinase different from PRK2. By molecular dynamic simulations, we show that new interactions between space-adjacent amino acid chains could be established by the presence of a di-anionic phosphate group on the analyzed serines to possibly modify RNA polymerase activity. Together, our data present novel evidence on the complex regulation at the finger subdomain of HCV NS5B via phosphorylation.
authors: Laboratorio de Virus Hepatitis, Departamento de Cs. Biológicas, Universidad Andrés Bello, Santiago, Chile.
authors: Sergio
authors: S
authors: Hernández
authors: Laboratorio de Virus Hepatitis, Departamento de Cs. Biológicas, Universidad Andrés Bello, Santiago, Chile.
authors: Daniella
authors: D
authors: Figueroa
authors: Centro de Bioinformática y Biología Integrativa, Facultad de Cs. Biológicas, Universidad Andrés Bello, Santiago, Chile.
authors: Simón
authors: S
authors: Correa
authors: Laboratorio de Virus Hepatitis, Departamento de Cs. Biológicas, Universidad Andrés Bello, Santiago, Chile.
authors: Ariel
authors: A
authors: Díaz
authors: Centro de Bioinformática y Biología Integrativa, Facultad de Cs. Biológicas, Universidad Andrés Bello, Santiago, Chile; Centro Interdisciplinario de Neurociencia de Valparaíso, Valparaíso, Chile.
authors: Daniel
authors: D
authors: Aguayo
authors: Laboratorio de Virus Hepatitis, Departamento de Cs. Biológicas, Universidad Andrés Bello, Santiago, Chile. Electronic address: rodrigo.villanueva@unab.cl.
authors: Rodrigo A
authors: RA
authors: Villanueva
mesh_terms: D002460:Cell Line; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D056004:Molecular Dynamics Simulation; D010766:Phosphorylation; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: Fingers; Hepatitis C virus; NS5B; Phosphorylation; RNA-dependent RNA polymerase