The hepatitis C virus NS2 protein generated by NS2-3 autocleavage is required for NS5A phosphorylation

Hepatitis C virus (HCV) is dependent on NS2-3 autocleavage and NS5A phosphorylation for its life cycle. We demonstrate that NS5A, when released from the NS2-5 polyprotein of the BK virus strain, is phosphorylated as two distinct forms, pp56 and pp58. Deletion analysis indicates that the appearance of pp58 requires NS2 in cis, while pp56 is NS2 independent. Disruption of NS2-3 autoproteolysis by directed mutagenesis results in loss of pp58. Expression of a construct producing NS2-3 is sufficient to restore pp58 in trans. These data indicate that generation of functional NS2 via autocleavage of the NS2-3 precursor and NS5A phosphorylation are consecutive processes, suggesting coordinate regulation during virus propagation in vivo.