Hepatitis CNS3 protease: Restoration of NS4A cofactor activity by N-biotinylation of mutated NS4A using synthetic peptides

The NS3 serine protase of Hepatitis C virus (HCV) requires NS4A protein as a cofactor for efficient cleavage at four sites in the nonstructural region . The cofactor activity has been mapped to the central hydrophobic region ( aa 22-34) of this 54-amino-acid NS4A protein, and site-directed mutagenesis has identified alternating hydrophobic amino acids, particularly Ile25 and Ile29, as critically important. A double mutant of NS4A cofactor peptide, I25A/I29A, completely abolished the cofactor activity. We now report that t he cofactor peptide activity in the I25A/I29A double mutant can be restored specifically by introducing a biotin-aminohexanoic acid fusion at the N-te rminus. In addition, a similar N-terminal fusion of biotin-aminohexanoic ac id with the wild-type 4A peptide sig -nificantly enhanced cofactor activity . Our data corroborate the crystal structure-based hypothesis of hydrophobi c interaction between the N-terminus of NS4A and the N-terminal alpha(0) he lix of NS3 protease. (C) 2000 Academic Press.