title: In vitro characterization of a purified NS2/3 protease variant of hepatitis C virus.
abstract: 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 protease is responsible for the cleavage of the downstream proteins. Purification and in vitro characterization of the NS2/3 protease has been hampered by its hydrophobic nature. NS2/3 protease activity could only be detected in cells or in in vitro translation assays with the addition of microsomal membranes or detergent. To facilitate purification of this poorly characterized 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 spans residues 904-1206 and includes the C-terminal half of NS2 and the N-terminal NS3 protease domain. The NS2/3 (904-1206) variant was purified from Escherichia coli inclusion bodies and refolded by gel filtration chromatography. The purified inactive form of NS2/3 (904-1206) was activated by the addition of glycerol and detergent to induce autocleavage at the predicted site 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 the cleavage site, or an N-terminal peptidic cleavage product. This NS2/3 variant will facilitate the development of an assay suitable for identifying inhibitors of HCV replication.
authors.affiliation: Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd., Research and Development, Laval, Québec H7S 2G5, Canada. dthibeault@lav.boehringer-ingelheim.com
authors.forename: D
authors.initials: D
authors.lastname: Thibeault
authors.forename: R
authors.initials: R
authors.lastname: Maurice
authors.forename: L
authors.initials: L
authors.lastname: Pilote
authors.forename: D
authors.initials: D
authors.lastname: Lamarre
authors.forename: A
authors.initials: A
authors.lastname: Pause
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D016174:Hepacivirus / Q000201:enzymology*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D017510:Protein Folding; D017361:Viral Nonstructural Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D017361:Viral Nonstructural Proteins; C097010:NS2 protein, Hepatitis C virus; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases