ACTIVITY OF PURIFIED HEPATITIS-C VIRUS PROTEASE NS3 ON PEPTIDE-SUBSTRATES

The protease domain of the hepatitis C virus (HCV) protein NS3 was exp ressed in Escherichia coli? purified to homogeneity, and shown to be a ctive on peptides derived from the sequence of the NS4A-NS4B junction. Experiments were carried out to optimize protease activity. Buffer re quirements included the presence of detergent, glycerol, and dithiothr eitol, pH between 7.5 and 8.5, and low ionic strength. C- and N-termin al deletion experiments defined a peptide spanning from the P6 to the P4' residue as a suitable substrate. Cleavage kinetics were subsequent ly measured by using decamer P6-P4' peptides corresponding to all inte rmolecular cleavage sites of the HCV polyprotein. The following order of cleavage efficiency, in terms of k(cat)/K-m, was determined: NS5A-N S5B > NS4A-NS4B much greater than NS4B-NS5A. A l-l-mer peptide contain ing residues 21 to 34 of the protease cofactor NS4A (Pep4A(21-34)), wh en added in stoichiometric amounts, was shown to increase cleavage rat es of all peptides, the largest effect (100-fold) being observed on th e hydrolysis of the NS4B-NS5A decamer. From the kinetic analysis of cl eavage data, we conclude that (i) primary structure is an important de terminant of the efficiency with which each site is cleaved during pol yprotein processing, (ii) slow cleavage of the NS4B-NS5A site in the a bsence of NS4A is due to low binding affinity of the enzyme for this s ite, and (iii) formation of a 1:1 complex between the protease and Pep 4A(21-34) is sufficient and required for maximum activation.