Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture

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.