Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B, and NS5A encoded on the same polyprotein

The nonstructural protein NS5A of hepatitis c virus (HCV) has been demonstr ated to be a phosphoprotein with an apparent molecular mass of 56 kDa. In t he presence of other viral proteins, p56 is converted into a slower-migrati ng form of NSSA (p58) by additional phosphorylation events. In this report, we show that the presence of NS3, NS4A, and NS4B together with NSSA is nec essary and sufficient for the generation of the hyperphosphorylated form of NSSA (p58) and that all proteins must be encoded on the same polyprotein ( in cis). Kinetic studies of NSSA synthesis and pulse-chase experiments demo nstrate that fully processed NS5A is the substrate for the formation of p58 and that p56 is converted to p58. To investigate the role of NS3 in NSSA h yperphosphorylation, point and deletion mutations were introduced into NS3 in the contest of a polyprotein containing the proteins from NS3 to NS5A. M utation of the catalytic serine residue into alanine abolished protease act ivity of NS3 and resulted in total inhibition of NS5A hyperphosphorylation, even if polyprotein processing was allowed by addition of NS3 and NS4A in trans. The same result was obtained by deletion of the first 10 or 28 N-ter minal amino acids of NS3, which are known to be important for the formation of a stable complex between NS3 and its cofactor NS4A. These data suggest that the formation of p58 is closely connected to HCV polyprotein processin g events. Additional data obtained with NS3 containing the 34 C-terminal re sidues of NS2 provide evidence that in addition to NS3 protease activity th e authentic N-terminal sequence is required for NSSA hyperphosphorylation.