ENHANCEMENT OF HEPATITIS-C VIRUS NS3 PROTEINASE ACTIVITY BY ASSOCIATION WITH NS4A-SPECIFIC SYNTHETIC PEPTIDES - IDENTIFICATION OF SEQUENCE AND CRITICAL RESIDUES OF NS4A FOR THE COFACTOR ACTIVITY

The NS3 proteinase of hepatitis C virus utilizes NS4A as a cofactor fo r cleavages at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the nonst ructural region of the viral polyprotein. To characterize NS4A for its role in modulating the NS3 proteinase activity at various cleavage si tes, synthetic peptides spanning various parts of NS4A were synthesize d and tested in a cell-free trans-cleavage reaction using purified NS3 proteinase domain and polyprotein substrates. The NS3 proteinase doma in was expressed in Escherichia coli, purified, denatured, and refolde d to an enzymatically active form. We found that a 12-amino-acid pepti de containing amino acid residues 22 to 33 in NS4A (CVVIVGRIVLSG) was sufficient for cofactor activity in NS3-mediated proteolysis. The pept ide enhanced the cleavage at the NS5A/5B site and was necessary for NS 3-mediated cleavage at NS4A/4B and NS4B/5A. Sequential amino acid subs titution within the designated peptide identified residues I-29 and I- 25 as critical for potential cofactor activity. We provide evidence th at the NS4A peptide and the NS3 catalytic domain form an enzymatically active complex. These data suggest that the central 12-amino-acid pep tide (aa 22-33) of NS4A is primarily important for the cofactor activi ty through complex formation with NS3, and the interaction may represe nt a new target for antiviral drug development. (C) 1996 Academic Pres s, Inc.