THE HEPATITIS-C VIRUS NS3 PROTEINASE - STRUCTURE AND FUNCTION OF A ZINC-CONTAINING SERINE PROTEINASE

The hepatitis C virus (HCV) NS3 protein contains a serine proteinase d omain implicated in the maturation of the viral polyprotein. NS3 forms a stable heterodimer with NS4A, a viral memebrane protein that acts a s an activator of the NS3 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 beta -strand contributed by NS4A is clamped between two beta-strands within the N terminus of NS3. Consistent with the requirement for extraordin arily long peptide substrates (P-6-P-4'), the structure of the NS3 pro teinase reveals a very long, solvent-exposed substrate-binding site. T he primary specificity pocket of the enzyme is shallow and closed at i ts bottm by Phe-154, explaining the preference of the NS3 proteinase f or cysteine residues in the substrate P-1 position. Another important feature of the NS3 proteinase is the presence of a tetrahedral zinc-bi nding 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. Inhibitio n of the NS3 proteinase activity is regarded as a promising strategy t o control the disease caused by HCV. Remarkably, the NS3 proteinase is susceptible to inhibition by the N-terminal cleavage products of subs trate peptides corresponding to the NS4A/NS4B, NS4B/NS5A and NS5A/NS5B cleavage sites. The K-i values of the inhibitory products are lower t han the K-m values of the respective substrates and follow the order N S4A<NS5A<NS4B. Starting from the observation that the NS3 proteinase u ndergoes product inhibition, very potent, active site-directed inhibit ors have been generated using a combinatorial peptide chemistry approa ch.