MECHANISTIC ROLE OF AN NS4A PEPTIDE COFACTOR WITH THE TRUNCATED NS3 PROTEASE OF HEPATITIS-C VIRUS - ELUCIDATION OF THE NS4A STIMULATORY EFFECT VIA KINETIC-ANALYSIS AND INHIBITOR MAPPING

Infection by hepatitis C viruses (HCVs) is a serious medical problem w ith no broadly effective treatment available for the progression of ch ronic hepatitis, The catalytic activity of a viral serine protease loc ated in the N-terminal one-third of nonstructural protein 3 (NS3) is r equired for polyprotein processing at four site-specific junctions, Th e three-dimensional crystal structure of the NS3-NS4A co-complex [Kim, J. L., Morgenstern, K. A., Lin, C. Fox, T., Dwyer, M. D., Landro, J. A. Chambers, S. P. Markland, W., Lepre, C, A., O'Malley, E. T., Harbes on, S. L., Rice, C. M., Murcko, M. A., Caron, P. R. & Thomson, J. A, ( 1996) Cell 87, 343-355] delineates a small hydrophobic region within t he 54-residue NS4A protein that intercalates with and makes extensive contacts to the core of the protease. The current investigation addres ses the mechanism of NS3 protease catalytic activation by NS4A utilizi ng a small synthetic NS4A peptide (residues 1678-1691 of the virus pol yprotein sequence) and the recombinantly expressed protease domain of NS3. The addition of NS4A dramatically increased NS3 k(cat) and k(cat) /K-m catalytic parameters when measured against small peptide substrat es representing the different site-specific junctions of the polyprote in. The catalytic effect of natural and non-natural amino acid substit utions at the P-1 position in a 5A/5B peptide substrate was investigat ed, NS3-NS4A demonstrated a marked catalytic preference for the cystei ne residue commonly found in authentic substrates. The pH dependence o f the NS3 hydrolysis reaction is not affected by the presence of NS4A. This result suggests that NS4A does not change the pK(a) values of th e active site residues of NS3 protease. A steady state kinetic analysi s was performed and indicated that the binding of NS4A and the peptide substrate occurs in an ordered fashion during the catalytic cycle, wi th NS4A binding first. Two distinct kinetic classes of peptidyl inhibi tors based upon the 5A/5B cleavage site were identified. An NS4A-indep endent class is devoid of prime residues. A second class of inhibitors is NS4A-dependent and contains a natural or non-natural cyclic amino acid substituted for the commonly found P-1' residue serine, These inh ibitors display an up to SO-fold increase in affinity for NS3 protease in the presence of NS4A. Sequential truncation of prime and P residue s from this inhibitor class demonstrated the face that the P-4' and P- 1' residues are crucial for potent inhibition. The selectivity of this NS4A effect is interpreted using a model of the 5A/5B decapeptide sub strate bound to the active site of the NS3-NS4A structure.