title: In vitro study of the NS2-3 protease of hepatitis C virus.
abstract: Processing at the C terminus of the NS2 protein of hepatitis C virus (HCV) is mediated by a virus-encoded protease which spans most of the NS2 protein and part of the NS3 polypeptide. In vitro cotranslational cleavage at the 2-3 junction is stimulated by the presence of microsomal membranes and ultimately results in the membrane insertion of the NS2 polypeptide. To characterize the biochemical properties of this viral protease, we have established an in vitro assay whereby the NS2-3 protease of HCV BK can be activated posttranslationally by the addition of detergents. The cleavage proficiency of several deletion and single point mutants was the same as that observed with microsomal membranes, indicating that the overall sequence requirements for proper cleavage at this site are maintained even under these artificial conditions. The processing efficiency of the NS2-3 protease varied according to the type of detergent used and its concentration. Also, the incubation temperature affected the cleavage at the 2-3 junction. The autoproteolytic activity of the NS2-3 protease could be inhibited by alkylating agents such as iodoacetamide and N-ethylmaleimide. Metal chelators such as EDTA and phenanthroline also inhibited the viral enzyme. The EDTA inhibition of NS2-3 cleavage could be reversed, at least in part, by the addition of ZnCl2 and CdCl2. Among the common protease inhibitors tested, tosyl phenylalanyl chloromethyl ketone and soybean trypsin inhibitor inactivated the NS2-3 protease. By means of gel filtration analysis, it was observed that the redox state of the reaction mixture greatly influenced the processing efficiency at the 2-3 site and that factors present in the rabbit reticulocyte lysate, wheat germ extract, and HeLa cell extract were required for efficient processing at this site. Thus, the in vitro assay should allow further characterization of the biochemical properties of the NS2-3 protease of HCV and the identification of host components that contribute to the efficient processing at the 2-3 junction.
authors.affiliation: I.R.B.M. Instituto di Ricerche di Biologia Molecolare P. Angeletti, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Pieroni
authors.forename: E
authors.initials: E
authors.lastname: Santolini
authors.forename: C
authors.initials: C
authors.lastname: Fipaldini
authors.forename: L
authors.initials: L
authors.lastname: Pacini
authors.forename: G
authors.initials: G
authors.lastname: Migliaccio
authors.forename: N
authors.initials: N
authors.lastname: La Monica
mesh_terms: D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation; D006367:HeLa Cells; D016174:Hepacivirus / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D017830:Octoxynol / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D003546:Cysteine Endopeptidases; D003902:Detergents; D017830:Octoxynol; D011092:Polyethylene Glycols; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; 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; C010615:Nonidet P-40; C107824:NS2-3 protease