Inhibition of hepatitis C virus NS2/3 processing by NS4A peptides - Implications for control of viral processing

The NS2/3 protease of hepatitis C virus is responsible for a single cleavag e in the viral polyprotein between the nonstructural proteins NS2 and NS3, The minimal protein region necessary to catalyze this cleavage includes mos t of NS2 and the N-terminal one-third of NS3, Autocleavage reactions using NS2/3 protein translated in vitro are used here to investigate the inhibito ry potential of peptides likely to affect the reaction, Peptides representi ng the cleaved sequence have no effect upon reaction rates, and the reactio n rate is insensitive to dilution, Both results are consistent with prior s uggestions that the NS2/3 cleavage is an intramolecular reaction. Surprisin gly, peptides containing the 12-amino acid region of NS4A responsible for b inding to NS3 inhibit the NS2/3 reaction with K-i values as low as 3 mu M. Unrelated peptide sequences of similar composition are not inhibitory, and neither are peptides containing incomplete segments of the NS4A region that binds to NS3, Inhibition of NS2/3 by NS4A peptides can be rationalized fro m the organizing effect of NS4A on the N terminus of NS3 (the NS2/3 cleavag e point) as suggested by the known three-dimensional structure of the NS3 p rotease domain. (Yan, Y., Li, Y., Munshi, S., Sardana, V., Cole, J. L., Sar dana, M., Steinkuhler, C., Tomei, L., De Francesco, R., Kuo, L. C., and Che n, Z. (1998) Protein Sci. 7, 837-847). These findings may imply a sequentia l order to proteolytic maturation events in hepatitis C virus.