HEPATITIS-C VIRUS-ENCODED NS2-3 PROTEASE - CLEAVAGE-SITE MUTAGENESIS AND REQUIREMENTS FOR BIMOLECULAR CLEAVAGE

Cleavage at the 2/3 site of hepatitis C virus (HCV) is thought to be m ediated by a virus-encoded protease composed of the region of the poly protein encoding NS2 and the N-terminal one-third of NS3. This proteas e is distinct from the NS3 serine protease, which is responsible for d ownstream cleavages in the nonstructural region. Site directed mutagen esis of residues surrounding the 2/3 cleavage site showed that cleavag e is remarkably resistant to single-amino-acid substitutions from P5 t o P3' (GWRLL down arrow API). The only mutations which dramatically in hibited cleavage were the ones most likely to alter the conformation d f the region, such as Pro substitutions at the P1 or P1' position, del etion of both amino acids at P1 and P1', or simultaneous substitution of multiple Ala residues. Cotransfection experiments were done to prov ide additional information on the polypeptide requirements for bimolec ular cleavage. Polypeptides used in these experiments contained amino acid substitutions and/or deletions in NS2 and/or the N-terminal one-t hird of NS3. Polypeptides with defects in either NS2 or the N-terminal portion of NS3 but not both were cleaved when cotransfected with cons tructs expressing intact versions of the defective region. Cotransfect ion experiments also showed that certain defective NS2-3 constructs pa rtially inhibited cleavage of wild-type polypeptides. Although these r esults show that inefficient cleavage can occur in a bimolecular react ion, they suggest that both molecules must contribute a functional sub unit to allow formation of a protease which is capable of cleavage at the 2/3 site. This reaction may resemble the cis cleavage thought to o ccur at the 2/3 site during processing of the wild-type HCV polyprotei n.