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J. Biol. Chem., Vol. 282, Issue 8, 5536-5544, February 23, 2007

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Hepatitis C Virus NS5A Is a Direct Substrate of Casein Kinase I-, a Cellular Kinase Identified by Inhibitor Affinity Chromatography Using Specific NS5A Hyperphosphorylation Inhibitors^*

From the Istituto di Ricerche di Biologia Molecolare "P. Angeletti," Via Pontina Km 30.6, 00040 Pomezia, Rome, Italy

The hepatitis C virus encodes a single polyprotein that is processed by host and viral proteases to yield at least 10 mature viral proteins. The nonstructural (NS) protein 5A is a phosphoprotein, and experimental data indicate that the phosphorylation state of NS5A is important for the outcome of viral RNA replication. We were able to identify kinase inhibitors that specifically inhibit the formation of the hyperphosphorylated form of NS5A (p58) in cells. These kinase inhibitors were used for inhibitor affinity chromatography in order to identify the cellular targets of these compounds. The kinases casein kinase I (CKI), p38 MAPK, CIT (Citron Rho-interacting kinase), GAK, JNK2, PKA, RSK1/2, and RIPK2 were identified in the high affinity binding fractions of two NS5A hyperphosphorylation inhibitors (NS5A-p58-i). Even though these kinases are targets of the NS5A-p58-i, the only kinase showing an effect on NS5A hyperphosphorylation was confirmed to be CKI-. Although this finding does not exclude the possibility that other kinase(s) might be involved in basal or regulatory phosphorylation of NS5A, we show here that NS5A is a direct substrate of CKI-. Moreover, in vitro phosphorylation of NS5A by CKI- resulted for the first time in the production of basal and hyperphosphorylated forms resembling those produced in cells. In vitro kinase reactions performed with NS5A peptides show that Ser-2204 is a preferred substrate residue for CKI- after pre-phosphorylation of Ser-2201.

Received for publication, November 10, 2006 , and in revised form, December 13, 2006.

^* 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.

¹ Present address: Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA 92037.

² To whom correspondence should be addressed. Tel.: 39-06-91093221; Fax: 39-06-91093654; E-mail: Petra_Neddermann{at}merck.com.

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