PHOSPHORYLATION OF THE HEPATITIS-C VIRUS NS5A PROTEIN IN-VITRO AND IN-VIVO - PROPERTIES OF THE NS5A-ASSOCIATED KINASE

NS5A derived from a hepatitis C virus (HCV) genotype Ib isolate has pr eviously been shown to undergo phosphorylation on serine residues (T, Kaneko, Y, Tanji, S. Satoh, M. Hijikata, S, Asabe, K, Kimura, and K, S himotohno, Biochem, Biophys, Res, Commun, 205:320-326, 1994), In this report, phosphorylation of NS5A derived from HCV isolates of the la an d distantly related 2a genotypes is demonstrated. Phosphoamino acid an alysis of NS5A from the la isolate indicated that phosphorylation occu rs predominantly on serine, with a minor fraction of threonine residue s also being phosphorylated, NS5A phosphorylation was observed in dive rse cell types, including COS-1, BHK-21, HeLa, and the hepatoma cell l ine HuH-7, Phosphorylation of a glutathione S-transferase (GST)/HCV-H NS5A fusion protein was also demonstrated in an in vitro kinase assay, This activity seemed to be highest when the pH of the reaction was ne utral or slightly alkaline and displayed a preference for Mn2+ over Mg 2+, with an optimum concentration of approximately 10 mM Mn2+, Somewha t surprisingly, in vitro phosphorylation of NS5A was inhibited by the addition of greater than or equal to 0.25 mM Ca2+ to reaction buffer c ontaining Mn2+ and/or Mg2+, Comparison of phosphopeptide maps of NS5A phosphorylated in vitro and in cultured cells showed that most of the phosphopeptides comigrated, suggesting that one or more kinases involv ed in NS5A phosphorylation in vivo and in vitro are the same, The effe cts of various kinase inhibitors on NS5A phosphorylation were consiste nt with a kinase activity belonging to the CMGC group of serine-threon ine kinases, The development of an in vitro kinase assay for NS5A phos phorylation should facilitate identification of kinase(s) responsible for its phosphorylation and of phosphorylation sites which may influen ce the function of NS5A in HCV propagation.