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

NS5A of the hepatitis C virus (HCV) is a highly phosphorylated protein involved in resistance against interferon and required most likely for replication of the viral genome. Phosphorylation of this protein is mediated by a cellular kinase(s) generating multiple proteins with different electrophoretic mobilities. In the case of the genotype 1b isolate HCV-J, in addition to the basal phosphorylated NS5A (designated pp56), a hyperphosphorylated form (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-length genomes of genotype 1b, competent or defective for NS5A hyperphosphorylation, we investigated the requirements for this NS5A modification. We found that hyperphosphorylation occurs when NS5A is expressed as part of a continuous NS3-5A polyprotein but not when it is expressed on its own or trans complemented with one or several other viral proteins. Results obtained with chimeras of both genomes show that single amino acid substitutions within NS3 that do not affect polyprotein cleavage can enhance or reduce NS5A hyperphosphorylation. Furthermore, mutations in the central or carboxy-terminal NS4A domain as well as small deletions in NS4B can also reduce or block hyperphosphorylation without affecting polyprotein processing. These requirements most likely reflect the formation of a highly ordered NS3-5A multisubunit complex responsible for the differential phosphorylation of NS5A and probably also for modulation of its biological activities.