METHOD FOR REPRODUCING IN VITRO THE PROTEOLYTIC ACTIVITY OF THE NS3 PROTEASE OF HEPATITIS C VIRUS (HCV)

摘要

This is a method for reproducing in vitro the serine protease activity associated with the HCV NS3 protein, that comprises the use both of sequences contained in NS3 and sequences contained in NS4A. This method takes advantage of the ability of the HCV NS4A protein, or sequences contained therein, to act as a cofactor of the serine protease activity or more generally of the enzymatic activities associated with NS3. Optimal serine protease activity is obtained when NS4A is present in a molar ratio of at least 1:1 with NS3. NS3 and NS4A can also be incorporated in the reaction mixture as NS3-NS4A precursor, as this precursor will generate, by means of an autoproteolytic event, equimolar amounts of NS3 and NS4A. It is also possible to mutate the cleavage site between NS3 and NS4A in a precursor, so that NS4A remains covalently bonded to NS3. The sequences that do not influence the proteolytic activity of NS3 can subsequently be removed from said non-proteolyzable precursor. The invention also relates to a composition of matter that comprises sequences contained in NS3 and NS4A, and to the use of these compositions for the setup of an enzymatic test capable of selecting, for therapeutic purposes, compounds that inhibit the enzymatic activity associated with NS3. The figure shows plasmidic vectors used in the method to activate HCV NS3 protease in cultivated cells and in vitro.