Effect of naturally occurring active site mutations on hepatitis C virus NS3 protease specificity

A comparison of the DNA sequences from all available genotypes of HCV indic ate that the active site residues of the NS3 protease are strictly conserve d with the exception of positions 123 and 168, which border the S-4 subsite , In genotype 3, the canonic arginine and aspartic acid have been replaced with threonine and glutamine, respectively, To determine if these differenc es contribute to an altered specificity, we characterized single chain NS3 proteases from strains la, Ib, and 3a with peptide substrates and product i nhibitors on the basis of the natural cleavage junction sequences, in addit ion to polyprotein substrates derived from the la strain. No statistically significant differences in specificity were observed. To demonstrate that t he active sites were actually different, we generated and evaluated peptide substrates with unnatural extended side-chains. These studies confirmed th at there are measurable differences between the NS3 proteases of genotypes 1 and 3, Specifically, a 5-fold difference in K-i was observed between the proteases from genotypes 1 and 3 when a D-Glu occupied P-5, and a 30-fold d ifference was seen when this position contained a D-homoglutamate. The cont ribution of residues 123 and 168 toward the altered specificity was then ev aluated individually by site directed mutagenesis, These mutants showed tha t potency differences within this series could be attributed to the residue that occupied position 123 of the protease. Modeling these unnatural subst rate/mutant protease interactions, on the basis of cocrystal structures of enzyme-substrate complexes, provides a structural basis for these observati ons. (C) 2001 Wiley-Liss, Inc.