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Please use this identifier to cite or link to this item: http://hdl.handle.net/2108/595

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contributor.advisorCesareni, Gianni-
contributor.advisorSantoro, Gabriella-
contributor.authorQuintavalle, Manuela-
description19. cicloen
description.abstractHepatitis C virus (HCV) has been the subject of intensive studies for nearly two decades. Nevertheless, some aspect of the virus life cycle are still a mystery. The HCV Nonstructural protein 5A (NS5A) has been shown to be a modulator of cellular processes possibly required for the establishment of viral persistence. NS5A is heavily phosphorylated, and a switch between a basally phosphorylated form of NS5A (p56) and a hyperphosphorylated form of NS5A (p58) seems to play a pivotal role in regulating HCV replication. Efficient replication of HCV subgenomic RNA in cell culture requires the introduction of adaptive mutations. Some of the most effective adaptive mutations occur at the serine residues that have been shown to be implicated in NS5A hyperphosphorylation and adaptive mutations at these sites result in a significant reduction of NS5A hyperphosphorylation (p58). After screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro, as well as the formation of NS5A p58 in cell culture. Cells transfected with the HCV wild type replicon sequence supported HCV RNA replication upon addition of any of the three compounds. Thus, reduction of the formation of p58 below a certain threshold either by adaptive mutations or by inhibition of the NS5A–specific kinase(s) would enable HCV replication in cell culture. Although large amounts of NS5A-p58 appear to inhibit HCV RNA replication, the complete inhibition of NS5A hyperphosphotylation by the kinase inhibitors we identified abolishes HCV replication of already adapted replicons indicating that a small quantity of p58 is required for replication. Using kinase inhibitors that specifically inhibit the formation of NS5A-p58 in cells, we identified CK1 kinase family as a target. NS5A-p58 increased upon overexpression of CK1alpha, CK1δ and CK1ε, whereas the RNA interference of only CK1alpha reduced NS5A hyperphosphorylation. Rescue of inhibition of NS5A-p58 was achieved by CK1 alpha overexpression, and we demonstrated that the CK1 alpha isoform is targeted by NS5A hyperphosphorylation inhibitors in living cells and that the down-regulation of NS5A attenuates HCV RNA replication. Finally, we demonstrate here that NS5A is a direct substrate of CKI- alpha and phosphorylation of NS5A in vitro by CKI- alpha resulted in the production of two phosphorylated forms that resemble those products produced in cells. In vitro kinase reactions performed with NS5A peptides show that S2204 is a preferred substrate residue for CKI- alpha after pre-phosphorylation of S2201en
format.extent3287567 bytes-
subject.classificationBIO/11 Biologia molecolareen
titleIdentification of cellular kinases responsible for Hepatitis C Virus NS5A hyperphosphorylationen
typeDoctoral thesisen
degree.nameDottorato in biologia cellulare e molecolareen
degree.disciplineFacoltà di Scienze Matematiche Fisiche e Naturalien
degree.grantorUniversità degli studi di Roma Tor Vergataen
date.dateofdefenseA.A. 2006/2007en
Appears in Collections:Tesi di dottorato in scienze matematiche e fisiche

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