ESTABLISHMENT OF AN IN-VITRO ASSAY TO CHARACTERIZE HEPATITIS-C VIRUS NS3-4A PROTEASE TRANS-PROCESSING ACTIVITY

An in vitro cleavage system was established to measure HCV NS3 proteas e trans-processing activity. This system utilizes purified NS3-4A prot ein from baculovirus, purified substrates expressed by in vitro transc ription and translation and defined buffer components. The 41-residue substrates, 5A/5B and 4A/4B, were processed efficiently in trans by wi ld-type NS3 but not by a catalytically inactive mutant protease; radio label sequencing confirmed that NS3-mediated cleavage occurred at the correct cysteine/serine sites, thereby authenticating this system. Two striking features of this in vitro assay are: (1) analogous 4B/5A and 3/4A substrates cannot be processed in trans under the same condition s, and (2) in vitro cleavage of the 5A/5B and 4A/4B sites is highly de pendent on the presence of NS4A, which we show is not the case in vivo .