Abstract
The NS2/3 protease of hepatitis C virus is responsible for a single cleavage in the viral polyprotein between the nonstructural proteins NS2 and NS3. The minimal protein region necessary to catalyze this cleavage includes most 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 inhibitory potential of peptides likely to affect the reaction. Peptides representing the cleaved sequence have no effect upon reaction rates, and the reaction rate is insensitive to dilution. Both results are consistent with prior suggestions that the NS2/3 cleavage is an intramolecular reaction. Surprisingly, peptides containing the 12-amino acid region of NS4A responsible for binding to NS3 inhibit the NS2/3 reaction with K(i) values as low as 3 microM. 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 from the organizing effect of NS4A on the N terminus of NS3 (the NS2/3 cleavage point) as suggested by the known three-dimensional structure of the NS3 protease domain (Yan, Y., Li, Y., Munshi, S., Sardana, V., Cole, J. L., Sardana, M., Steinkuhler, C., Tomei, L., De Francesco, R., Kuo, L. C., and Chen, Z. (1998) Protein Sci. 7, 837-847). These findings may imply a sequential order to proteolytic maturation events in hepatitis C virus.
Highlights
The NS2/3 protease of hepatitis C virus is responsible for a single cleavage in the viral polyprotein between the nonstructural proteins NS2 and NS3
The Hepatitis C virus (HCV) genome encodes a single polyprotein of approximately 3000 amino acids containing the viral proteins in the following order: C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B
Release of mature NS4A, NS4B, NS5A, and NS5B is catalyzed by the chymotrypsin-like serine protease contained within the N-terminal domain of NS3, whereas host cell proteases release C, E1, E2, and p7, creating the N terminus of NS2 at amino acid 810 [10, 11]
Summary
The NS2/3 protease of hepatitis C virus is responsible for a single cleavage in the viral polyprotein between the nonstructural proteins NS2 and NS3. Inhibition of NS2/3 by NS4A peptides can be rationalized from the organizing effect of NS4A on the N terminus of NS3 (the NS2/3 cleavage point) as suggested by the known three-dimensional structure of the NS3 protease domain The HCV genome encodes a single polyprotein of approximately 3000 amino acids containing the viral proteins in the following order: C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. Considering the paucity of data regarding the biochemistry of the NS2/3 reaction, it is of interest to probe the effects of peptides that represent natural ligands of NS2/3 upon the NS2/3 reaction rate These peptides include sequences containing the NS3 binding region of NS4A as well as sequences containing the NS2/3 cleavage site. The unexpected result of this study is that NS4A is a potent inhibitor, whereas the reaction is unaffected by potentially competing cleavage-site peptides
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