The Hepatitis C Virus NS3 Proteinase: Structure and Function of a Zinc-Containing Serine Proteinase

Abstract

The hepatitis C virus (HCV) NS3 protein contains a serine proteinase domain implicated in the maturation of the viral polyprotein. NS3 forms a stable heterodimer with NS4A, a viral memebrane protein that acts as an activator of the IMS3 proteinase. The three-dimensional structure of the NS3 proteinase complexed with an NS4A-derived peptide has been determined. The NS3 proteinase adopts a chymotrypsin-like fold. A β-strand contributed by NS4A is clamped between two β-strands within the N terminus of NS3. Consistent with the requirement for extraordinarily long peptide substrates (P₆-P₄’), the structure of the NS3 proteinase reveals a very long, solvent-exposed substrate-binding site. The primary specificity pocket of the enzyme is shallow and closed at its bottm by Phe-154, explaining the preference of the NS3 proteinase for cysteine residues in the substrate P, position. Another important feature of the NS3 proteinase is the presence of a tetrahedral zinc-binding site formed by residues Cys-97, Cys-99, Cys-145 and His-149. The zinc-binding site has a role in maintaining the structural stability and guiding the folding of the NS3 serine proteinase domain. Inhibition of the NS3 proteinase activity is regarded as a promising strategy to control the disease caused by HCV. Remarkably, the NS3 proteinase is susceptible to inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A/NS4B, NS4B/NS5A and NS5A/NS5B cleavage sites. The K_i values of the inhibitory products are lower than the K_m values of the respective substrates and follow the order NS4A<NS5A<NS4B. Starting from the observation that the NS3 proteinase undergoes product inhibition, very potent, active site-directed inhibitors have been generated using a combinatorial peptide chemistry approach.