Mechanism of Dissociative Inhibition of HIV Protease and Its Autoprocessing from a Precursor

Dimerization is indispensible for release of the human immunodeficiency virus protease (PR) from its precursor (Gag-Pol) and ensuing mature-like catalytic activity that is crucial for virus maturation. We show that a single-chain Fv fragment (scFv) of a previously reported monoclonal antibody (mAb1696), which recognizes the N-terminus of PR, dissociates a dimeric mature D25N PR mutant with an enhanced dimer dissociation constant (Kd) in the sub-micromolar range to form predominantly a monomer–scFv complex at a 1:1 ratio, along with small (5–10%) amounts of a dimer–scFv complex. Enzyme kinetics indicate a mixed mechanism of inhibition of the wild-type PR, which exhibits a Kd < 10 nM, with effects both on Km and kcat at an scFv-to-PR ratio of 10:1. ScFv binds to the N-terminal peptide P1QITLW6 of PR and to PR monomers with dissociation constants of ≤ 30 nM and ~ 100 nM, respectively. Consistent with an ~ 400-fold increase in the dissociation of the antibody (KAb) on even addition of an acetyl group to P1 of the peptide, the antibody fails to inhibit N-terminal autoprocessing of the PR from a model precursor (at ~ 5 μM). However, subsequent to this cleavage, it sequesters the PR, thus blocking autoprocessing at its C-terminus. A second monoclonal antibody [PRM1 (human monoclonal antibody to PR)], which recognizes part of the flap region (residues 41–47) of the mature PR and its precursor, does not inhibit autoprocessing and ensuing catalytic activity. However, its failure to recognize drug-resistant clinical mutants of PR may be beneficial to monitor the selection of mutations in this region under drug pressure.