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J. Biol. Chem., Vol. 279, Issue 48, 50031-50041, November 26, 2004

This Article Full Text Full Text (PDF) All Versions of this Article: 279/48/50031    most recent M408617200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, S.-J. Articles by Oh, J.-W. Search for Related Content PubMed PubMed Citation Articles by Kim, S.-J. Articles by Oh, J.-W. Social Bookmarking                      What's this?

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, and Jong-Won Oh

From the Department of Biotechnology, Yonsei University, Seoul 120-749, Korea

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.

Received for publication, July 29, 2004

^* * This work was supported by Korea Research Foundation Grant KRF-2003-015-C00492. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Biotechnology, Yonsei University, 134 Sinchon-dong, Seodaemun-gu, Seoul 120-749, Korea. Tel.: 82-2-2123-2881; Fax: 82-2-362-7265; E-mail: jwoh{at}yonsei.ac.kr.

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