Novel macromolecular inhibitors of human immunodeficiency virus-1 protease

Abstract

An intracellularly expressed defective human immunodeficiency virus type-1 (HIV-1) protease (PR) monomer could function as a dominant-negative inhibitor of the enzyme that requires dimerization for activity. Based on in silico studies, two mutant PRs harboring hydrophilic mutations, capable of forming favorable inter- and intra-subunit interactions, were selected: PR_RE containing Asp25Arg and Gly49Glu mutations, and PR_RER containing an additional Ile50Arg mutation. The mutants were expressed and tested by PR assays, nuclear magnetic resonance (NMR) and cell culture experiments. The mutant PRs showed dose-dependent inhibition of the wild-type PR in a fluorescent microtiter plate PR assay. Furthermore, both mutants were retained by hexahistidine-tagged wild-type HIV-1 PR immobilized on nickel-chelate affinity resin. For the first time, heterodimerization between wild-type and dominant-negative mutant PRs were also demonstrated by NMR spectroscopy. ¹H–¹⁵N Heteronuclear Single Quantum Coherence NMR spectra showed that although PR_RE has a high tendency to aggregate, PR_RER exists mainly as a folded monomer at 25–35 µM concentration, but in the presence of wild-type PR in a ratio of 1:1, heterodimerization occurs with both mutants. While the recombinant virus containing the PR_RE sequence showed only very low level of expression, expression of the viral proteins of the virus with the PR_RER sequence was comparable with that of the wild-type. In cell culture experiments, infectivity of viral particles containing PR_RER protein was reduced by 82%, at mutant to wild-type infective DNA ratio of 2:1.