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J. Biol. Chem., Vol. 283, Issue 1, 29-36, January 4, 2008

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Identification of Human Kinases Involved in Hepatitis C Virus Replication by Small Interference RNA Library Screening^*

Lubica Supekova, Frantisek Supek, Jongkook Lee, Shawn Chen, Nathanael Gray^¶, John P. Pezacki^||, Achim Schlapbach^**, and Peter G. Schultz¹

From the Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, Department of Drug Discovery, Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, ^¶Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Dana Farber Cancer Institute, Boston, Massachusetts 02115, ^||Steacie Institute for Molecular Sciences Ottawa, Ontario K1A 0R6, Canada, and ^**Novartis Institute for Biomedical Research, Basel CH-4002, Switzerland

The propagation of the hepatitis C virus (HCV) is a complex process that requires both host and viral proteins. To facilitate identification of host cell factors that are required for HCV replication, we screened a panel of small interference RNAs that preferentially target human protein kinases using an HCV replicon expressing the firefly luciferase gene as a genetic reporter. Small interference RNAs specific for three human kinases, Csk, Jak1, and Vrk1, were identified that reproducibly reduce viral RNA and viral protein levels in HCV replicon-bearing cells. Treatment of replicon cells with a small molecule inhibitor of Csk also resulted in a significant reduction in HCV RNA and proteins, further supporting a role for Csk in HCV replication. The effects of siRNAs targeting eight kinases known to be negatively regulated by Csk were then examined; knock down of one of these kinases, Fyn, resulted in up-regulation of the HCV replicon, suggesting that Csk mediates its effect on HCV replication through Fyn. This conclusion was further corroborated by demonstration that replicon cells treated with Csk inhibitor contained lower levels of the phosphorylated form of Fyn than control cells.

Received for publication, May 15, 2007 , and in revised form, October 17, 2007.

^* 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.

¹ To whom correspondence should be addressed: 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-9300; Fax: 858-784-9440; E-mail: schultz{at}scripps.edu.

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