SERINE-PROTEASE OF HEPATITIS-C VIRUS EXPRESSED IN INSECT CELLS AS THENS3 4A COMPLEX/

Hepatitis C virus (HCV) protease NS3 and its protein activator NS4A pa rticipate in the processing of the viral polyprotein into its constitu ent nonstructural proteins. The NS3/4A complex is thus an attractive t arget for antiviral therapy against HCV. We expressed the full-length NS3 and NS4A in insect cells as a soluble fusion protein with an N-ter minal polyhistidine tag and purified the two proteins to homogeneity. Cleavage at the junction between HisNS3 and NS4A occurs during express ion, producing a noncovalent complex between HisNS3 and NS4A with a su bnanomolar dissociation constant. We purified the HisNS3/4A complex by detergent extraction of cell lysate and by metal chelate chromatograp hy. We removed the His tag by thrombin cleavage and then further purif ied the complex by gel filtration. The purified NS3/4A complex is acti ve in a protease assay using a synthetic peptide substrate derived fro m the NS5A-NS5B junction, with k(cat)/K-m of 3700 (+/- 600) M-1 s(-1), an order of magnitude above those previously reported for NS3 express ed by other strategies. This high protease activity implies that the f ull-length sequences of NS3 and NS4A are required for optimal activity of the NS3 protease domain. We examined the dependence of the NS3/4A protease activity on buffer conditions, temperature, and the presence of detergents. We find that, under most conditions, NS3 protease activ ity is dependent on the aggregation state of the NS3/4A complex. The m onodisperse, soluble form of the NS3/4A complex is associated with the highest protease activity.