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.