ANALYSIS OF NS3-MEDIATED PROCESSING OF THE HEPATITIS-C VIRUS NONSTRUCTURAL REGION IN-VITRO

The protease activity of the hepatitis C virus (HCV) NS3 protein has b een investigated using transient expression methods in mammalian cells , as well as in vitro transcription/translation systems. We confirmed that expression of the NS3-5 polyprotein in rabbit reticulocyte lysate s results in efficient cis processing at the NS3/NS4 junction. However , processing at the other predicted sites of NS3-mediated cleavage var ied markedly in efficiency, the site most susceptible being that betwe en NS5A and NS5B. Time-course analysis of the proteolytic processing o f the HCV non-structural precursor showed that the cis cleavage betwee n NS3 and NS4 occurred extremely rapidly. However, efficient cleavage at this position was dependent on the prior removal of the NS2 protein . Furthermore, the presence of uncleaved NS2 sequences on the enzyme s everely impeded NS3-mediated proteolysis at downstream sites in the po lyprotein. This suggests therefore that efficient cleavage at the NS2/ NS3 junction is a pivotal event in HCV replication. During the course of this study a proteolytically inactive mutant of NS3 was characteriz ed carrying a previously unreported amino acid substitution near the p roposed active site of the enzyme. Molecular modelling suggested that the amino acid present at this position may influence the conformation of the active site of the enzyme. Recently a number of reports have d escribed a second protease activity, located in the NS2/NS3 region, wh ich is responsible for cleavage at the NS2/NS3 junction. We have ident ified an isolate of HCV, obtained from a U.K. patient, which has a vir tually inactive NS2/NS3 protease. The possible implications of this ob servation are discussed.