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. (1)H-(15)N 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 microM 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.

Inhibition Profiling of Retroviral Protease Inhibitors Using an HIV-2 Modular System

“…In accordance with an HIV-1 transfection protocol [ 52 ], 293T human embryonic kidney cells (Invitrogen) were seeded in T75 flask in 15 mL Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), 1% glutamine and 1% penicillin-streptomycin. The day before the transfection, cells were passaged in order to achieve 70% confluency the next day.…”

Analysis of networks of host proteins in the early time points following HIV transduction

“…Viral particles were produced with some modifications of a previously utilized protocol [48]. Briefly, recombinant viruses were produced by transient transfection of 293 T cells (ATCC® CRL3216™) using pWOX-CMV-GFP (transfer vector plasmid), pMDLg/pRRE (packaging plasmid), pRSV.rev (Rev-coding plasmid), and pMD.…”

“…G (VSV-G envelope protein-coding plasmid). Vectors were a kind gift from D. Trono (University of Geneva Medical School, Geneva, Switzerland) [49], and were subsequently modified by our research group [48]. Salmon sperm DNA (Sigma-Aldrich) was also added.…”

Understanding HIV-1 Protease Autoprocessing for Novel Therapeutic Development

“…The catalytic site mutation D25N alone increased the equilibrium dimer dissociation constant by a factor >100-fold (1.3 ± 0.09 μM) relative to the wild-type mature PR [47]. Other residues including D25, I49, G50 at the interface were also exploited to promote the formation of heterodimers between the wild-type PR and the inactive mutant [104,105]. Interestingly, when introduced into the provirus and mixed with the wild-type provirus DNA at ratio of 2:1, the resulting mutant reduced virus infectivity by 82%, providing a proof-of-concept to inhibit HIV replication via manipulating PR dimer interface.…”