Protein kinase C-related kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation

Seong Jun Kim, Jung Hee Kim, Yeon Gu Kim, Ho Soo Lim, Jong-Won Oh

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

The hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase required for replication of the HCV RNA genome. We have identified a peptide that most closely resembles a short region of the protein kinase C-related kinase 2 (PRK2) by screening of a random 12-mer peptide library displayed on the surface of the M13 bacteriophage with NS5B proteins immobilized on microwell plates. Competitive phage enzyme-linked immunosorbent assay with a synthetic peptide showed that the phage clone displaying this peptide could bind HCV RNA polymerase with a high affinity. Coimmunoprecipitation and colocalization studies demonstrated in vivo interaction of NS5B with PRK2. In vitro kinase assays demonstrated that PRK2 specifically phosphorylates NS5B by interaction with the N-terminal finger domain of NS5B (amino acids 1-187). Consistent with the in vitro NS5B-phosphorylating activity of PRK2, we detected the phosphorylated form of NS5B by metabolic cell labeling. Furthermore, HCV NS5B immunoprecipitated from HCV subgenomic replicon cells was specifically recognized by an antiphosphoserine antibody. Knock-down of the endogenous PRK2 expression using a PRK2-specific small interfering RNA inhibited HCV RNA replication. In contrast, PRK2 overexpression, which was accompanied by an increase of in the level of its active form, dramatically enhanced HCV RNA replication. Altogether, our results indicate that HCV RNA replication is regulated by NS5B phosphorylation by PRK2.

Original languageEnglish
Pages (from-to)50031-50041
Number of pages11
JournalJournal of Biological Chemistry
Volume279
Issue number48
DOIs
Publication statusPublished - 2004 Nov 26

Fingerprint

Phosphorylation
DNA-Directed RNA Polymerases
Viruses
Hepacivirus
Protein Kinase C
Phosphotransferases
Bacteriophages
Virus Replication
RNA
Peptides
Assays
Immobilized Proteins
Bacteriophage M13
RNA Replicase
Peptide Library
Replicon
protein kinase C kinase
Immunosorbents
Viral RNA
Small Interfering RNA

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Kim, Seong Jun ; Kim, Jung Hee ; Kim, Yeon Gu ; Lim, Ho Soo ; Oh, Jong-Won. / Protein kinase C-related kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 48. pp. 50031-50041.
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Protein kinase C-related kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation. / Kim, Seong Jun; Kim, Jung Hee; Kim, Yeon Gu; Lim, Ho Soo; Oh, Jong-Won.

In: Journal of Biological Chemistry, Vol. 279, No. 48, 26.11.2004, p. 50031-50041.

Research output: Contribution to journalArticle

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