Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructuralproteins NS3, NS4A, and NS4B

NS5A of the hepatitis C virus (HCV) is a highly phosphorylated protein invo lved in resistance against interferon and required most likely for replicat ion of the viral genome. Phosphorylation of this protein is mediated by a c ellular kinase(s) generating multiple proteins with different electrophoret ic mobilities. In the case of the genotype 1b isolate HCV-J, in addition to the basal phosphorylated NS5A (designated pp56), a hyperphosphorylated for m (pp58) was found on coexpression of NS4A (T. Kaneko, Y. Tanji, S. Satoh, M. Hijikata, S. Asabe, K. Kimura, and K. Shimotohno, Biochem. Biophys. Res. Commun. 205:320-326, 1994), Using a comparative analysis of two full-lengt h genomes of genotype 1b, competent or defective for NS5A hyperphosphorylat ion, we investigated the requirements for this NS5A modification. We found that hyperphosphorylation occurs when NS5A is expressed as part of a contin uous NS3-5A polyprotein but not when it is expressed on its own or trans co mplemented with one or several other viral proteins. Results obtained with chimeras of both genomes show that single amino acid substitutions within N S3 that do not affect polyprotein cleavage can enhance or reduce NS5A hyper phosphorylation. Furthermore, mutations in the central or carboxy-terminal NS4A domain as well as small deletions in NS4B can also reduce or block hyp erphosphorylation without affecting polyprotein processing. These requireme nts most likely reflect the formation of a highly ordered NS3-5A multisubun it complex responsible for the differential phosphorylation of NS5A and pro bably also for modulation of its biological activities.