In vitro characterization of a purified NS2/3 protease variant of hepatitis C virus.

The cleavage of the hepatitis C virus polyprotein between the nonstructural proteins NS2 and NS3 is mediated by the NS2/3 protease, whereas the NS3 pr otease is responsible for the cleavage of the downstream proteins. Purifica tion and in vitro characterization of the NS2/3 protease has been hampered by its hydrophobic nature. NS2/3 protease activity could only be detected i n cells or in in vitro translation assays with the addition of microsomal m embranes or detergent. To facilitate purification of this poorly characteri zed protease, we truncated the N-terminal hydrophobic domain, resulting in an active enzyme with improved biophysical properties. We define a minimal catalytic region of NS2/3 protease retaining autocleavage activity that spa ns residues 904-1206 and includes the C-terminal half of NS2 and the N-term inal NS3 protease domain. The NS2/3 (904-1206) variant was purified from Es cherichia coli inclusion bodies and refolded by gel filtration chromatograp hy. The purified inactive form of NS2/3 (904-1206) was activated by the add ition of glycerol and detergent to induce autocleavage at the predicted sit e between Leu(1026) and Ala(1027). NS2/3 (904-1206) activity was dependent on zinc ions and could be inhibited by NS4A peptides, peptides that span th e cleavage site, or an N-terminal peptidic cleavage product. This NS2/3 var iant will facilitate the development of an assay suitable for identifying i nhibitors of HCV replication.