title: Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture.
abstract: Efficient replication of hepatitis C virus (HCV) subgenomic RNA in cell culture requires the introduction of adaptive mutations. In this report we describe a system which enables efficient replication of the Con1 subgenomic replicon in Huh7 cells without the introduction of adaptive mutations. The starting hypothesis was that high amounts of the NS5A hyperphosphorylated form, p58, inhibit replication and that reduction of p58 by inhibition of specific kinase(s) below a certain threshold enables HCV replication. Upon screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro and the formation of NS5A p58 in cell culture. Cells, transfected with the HCV Con1 wild-type sequence, support HCV RNA replication upon addition of any of the three compounds. The effect of the kinase inhibitors was found to be synergistic with coadaptive mutations in NS3. This is the first direct demonstration that the presence of high amounts of NS5A-p58 causes inhibition of HCV RNA replication in cell culture and that this inhibition can be relieved by kinase inhibitors.
authors.affiliation: IRBM, Via Pontina Km 30,600, 00040 Pomezia, Rome, Italy. Petra_Neddermann@merck.com
authors.forename: Petra
authors.initials: P
authors.lastname: Neddermann
authors.forename: Manuela
authors.initials: M
authors.lastname: Quintavalle
authors.forename: Chiara
authors.initials: C
authors.lastname: Di Pietro
authors.forename: Angelica
authors.initials: A
authors.lastname: Clementi
authors.forename: Mauro
authors.initials: M
authors.lastname: Cerretani
authors.forename: Sergio
authors.initials: S
authors.lastname: Altamura
authors.forename: Linda
authors.initials: L
authors.lastname: Bartholomew
authors.forename: Raffaele
authors.initials: R
authors.lastname: De Francesco
mesh_terms: D002478:Cells, Cultured; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D012367:RNA, Viral / Q000096:biosynthesis*; D012093:Replicon; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
chemical_list: D011494:Protein Kinases; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase