IN-VITRO STUDIES ON THE ACTIVATION OF THE HEPATITIS-C VIRUS NS3 PROTEINASE BY THE NS4A COFACTOR

Proteolytic processing of the nonstructural proteins of the hepatitis C virus (HCV) is mediated by two viral proteinases: the NS2-3 proteina se cleaving at the NS2/3 junction and the NS3 serine-type proteinase r esponsible for processing at the NS3/4A, NS4A/B, NS4B/5A, and NSSA/B s ites. Activity of the NS3 proteinase is modulated by NS4A. In the abse nce of this cofactor processing al the NS3-dependent sites does not oc cur or, in the case of the NSSA/B junction, is poor but increased when NS4A is present. Although recent studies demonstrated that proteinase activation requires direct interaction between NS3 and NS4A, the mech anism by which NS4A exerts the activation function is not known. To fu rther analyze the conditions of proteinase activation and to character ize the NS3 sequences important for complex formation and activation w e used an in vitro assay in which radiolabeled HCV substrates were mix ed with NS3 proteinase and synthetic NS4A peptides. We found that micr osomal membranes are not required for proteinase activation, However, they are important for efficient accessibility of the NS4A/B site but not the other trans-cleavage sites. Studies with NS3 deletion mutants identified a region between amino acids 15 and 22 which is essential f or proteinase activation. Results obtained with several mutations intr oduced into this sequence show that a weak overall association between NS3 and NS4A is sufficient for proteinase activation and suggest that a beta-sheet at the NS3 amino terminus plays an important role. Altho ugh not essential for proteinase activation the amino terminal 14 NS3 residues were found to have an auxilliary function probably by stabili zing the NS3/4A interaction. Finally, we could demonstrate intracellul ar, peptide-mediated modulation of proteinase activity providing the b asis for the development of a novel therapeutic concept. (C) 1996 Acad emic Press, Inc