IN-VITRO STUDY OF THE NS2-3 PROTEASE OF HEPATITIS-C VIRUS

Citation
L. Pieroni et al., IN-VITRO STUDY OF THE NS2-3 PROTEASE OF HEPATITIS-C VIRUS, Journal of virology, 71(9), 1997, pp. 6373-6380
Citations number
34
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Virology
Journal title
ISSN journal
0022-538X
Volume
71
Issue
9
Year of publication
1997
Pages
6373 - 6380
Database
ISI
SICI code
0022-538X(1997)71:9<6373:ISOTNP>2.0.ZU;2-D
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 N S2 protein and part of the NS3 polypeptide. In vitro cotranslational c leavage at the 2-3 junction is stimulated by the presence of microsoma l membranes and ultimately results in the membrane insertion of the NS 2 polypeptide. To characterize the biochemical properties of this vira l protease, we have established an in vitro assay whereby the NS2-3 pr otease 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 cleavag e at this site are maintained even under these artificial conditions. The processing efficiency of the NS2-3 protease varied according to th e type of detergent used and its concentration. Also, the incubation t emperature affected the cleavage at the 2-3 junction. The autoproteoly tic activity of the NS2-3 protease could be inhibited hy alkylating ag ents such as iodoacetamide and N-ethylmaleimide. Metal chelators such as EDTA and phenanthroline also inhibited the viral enzyme. The EDTA i nhibition of NS2-3 cleavage could be reversed, at least in part, by th e addition of ZnCl2, and CdCl2. Among the common protease inhibitors t ested, tosyl phenylalanyl chloromethyl ketone and soybean trypsin inhi bitor inactivated the NS2-3 protease. By means of gel filtration analy sis, it was observed that the redox state of the reaction mixture grea tly influenced the processing efficiency at the 2-3 site and that fact ors present in the rabbit reticulocyte lysate, wheat germ extract, and HeLa cell extract were required far efficient processing at this site . Thus, the in vitro assay should allow further characterization of th e biochemical properties of the NS2-3 protease of HCV and the identifi cation of host components that contribute to the efficient processing at the 2-3 junction.