Phosphorylation at the N-terminal finger subdomain of a viral RNA-dependent RNA polymerase

Sergio Hernández, Daniella Figueroa, Simón Correa, Ariel Díaz, Daniel Aguayo, Rodrigo A. Villanueva

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish
Article number34455
Pages (from-to)21-27
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume466
Issue number1
DOIs
Publication statusPublished - 19 Aug 2015

Keywords

  • Fingers
  • Hepatitis C virus
  • NS5B
  • Phosphorylation
  • RNA-dependent RNA polymerase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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