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

Resultado de la investigación: Article

1 Cita (Scopus)

Resumen

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.

Idioma originalEnglish
Número de artículo34455
Páginas (desde-hasta)21-27
Número de páginas7
PublicaciónBiochemical and Biophysical Research Communications
Volumen466
N.º1
DOI
EstadoPublished - 19 ago 2015

Huella dactilar

RNA Replicase
Phosphorylation
Viral RNA
Viruses
Fingers
Phosphotransferases
Genotype
Hepacivirus
Serine
Phosphopeptides
Bioelectric potentials
DNA-Directed RNA Polymerases
Bioinformatics
RNA Polymerase III
Protein Kinase C
Mass spectrometry
Molecular dynamics
Assays
Substitution reactions
Molecular Dynamics Simulation

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Citar esto

Hernández, Sergio ; Figueroa, Daniella ; Correa, Simón ; Díaz, Ariel ; Aguayo, Daniel ; Villanueva, Rodrigo A. / Phosphorylation at the N-terminal finger subdomain of a viral RNA-dependent RNA polymerase. En: Biochemical and Biophysical Research Communications. 2015 ; Vol. 466, N.º 1. pp. 21-27.
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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.",
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Phosphorylation at the N-terminal finger subdomain of a viral RNA-dependent RNA polymerase. / Hernández, Sergio; Figueroa, Daniella; Correa, Simón; Díaz, Ariel; Aguayo, Daniel; Villanueva, Rodrigo A.

En: Biochemical and Biophysical Research Communications, Vol. 466, N.º 1, 34455, 19.08.2015, p. 21-27.

Resultado de la investigación: Article

TY - JOUR

T1 - Phosphorylation at the N-terminal finger subdomain of a viral RNA-dependent RNA polymerase

AU - Hernández, Sergio

AU - Figueroa, Daniella

AU - Correa, Simón

AU - Díaz, Ariel

AU - Aguayo, Daniel

AU - Villanueva, Rodrigo A.

PY - 2015/8/19

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AB - 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.

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