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

Manuela Quintavalle,Sonia Sambucini,Vincenzo Summa,Laura Orsatti,Fabio Talamo,Raffaele De Francesco,Petra Neddermann

doi:10.1074/jbc.m610486200

Abstract

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-alpha. 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-alpha. Moreover, in vitro phosphorylation of NS5A by CKI-alpha 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-alpha after pre-phosphorylation of Ser-2201.

Highlights

The 9.6-kb RNA genome of hepatitis C virus (HCV) is translated into a single polyprotein, which is co- and post-translationally processed by host and viral proteases to yield at least 10 mature viral proteins (C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B) [1]
We have introduced the S2204R mutation in NS5A, and the effect on NS5A hyperphosphorylation is clearly visible when expressed from the HCV polyprotein in cells
We have demonstrated for the first time that casein kinase I (CKI)-␣ is directly involved in NS5A hyperphosphorylation in cells [24]

Summary

Introduction

The 9.6-kb RNA genome of HCV is translated into a single polyprotein, which is co- and post-translationally processed by host and viral proteases to yield at least 10 mature viral proteins (C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B) [1]. This might result in a less tight interaction with membranes, and dissociation of NS5A from the replication complex might be facilitated In support of this hypothesis are recent publications that demonstrate that mutations within NS5A can be transcomplemented, whereas trans-complementation for all other nonstructural proteins failed [6, 7]. The requirements for NS5A hyperphosphorylation are in remarkable agreement with two other previous observations as follows: trans-complementation could be obtained only when NS5A was expressed in the context of an intact polyprotein coding from NS3 to NS5A [6], and wild type NS5A exerted a trans-dominant negative effect on adapted subgenomic replicons only when expressed in the same context [17] It seems that the presence of the NS3, NS4A, and NS4B proteins. Whether NS5A hyperphosphorylation is the cause or the consequence of correct folding in this context is still to be established

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