IN-VITRO ACTIVITY OF HEPATITIS-C VIRUS PROTEASE NS3 PURIFIED FROM RECOMBINANT BACULOVIRUS-INFECTED SF9 CELLS

A recombinant Baculovirus expression system was used for the productio n of a 20-kDa protein encompassing the hepatitis C virus NS3 protease domain. The protein was purified to apparent homogeneity after deterge nt extraction of cell homogenates. It was shown to be a monomer in sol ution and to cleave the in vitro translated precursor proteins NS4A-NS 4B and NS5A-N95B, but not the NS4B-NS5A or the NS3-NS4A precursors, Th e enzyme also cleaved a 20-mer peptide corresponding to the NS4A-NS4B junction with k(cat)/K-m = 174 M(-1) s(-1). Peptides harboring NS4A se quences comprising amino acids 21-54 (Pep4A(21-54)) and 21-34 (Pep4A(2 1-34)) were found to induce an up to 2.8-fold acceleration of cleavage , Kinetic analysis revealed that this acceleration was due to an incre ase in k(cat), whereas no significant effect on K-m could be detected, Pep4A(21-54) was also an absolute requirement for cleavage of in vitr o translated NS4B-NS5A by the purified protease, From these data we co nclude that: (i) the purified protease domain shows substrate specific ity and cleavage requirements similar to those previously reported on the basis of transfection experiments, (ii) activation of the purified protease by the NS4A co-factor can be mimicked by synthetic peptide a nalogs, and (iii) a central hydrophobic region of NS4A with a minimum core of 14 amino acids is responsible for the interaction with NS3.