Potent Nonnucleoside Reverse Transcriptase Inhibitors Target HIV-1 Gag-Pol

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) target HIV-1 reverse transcriptase (RT) by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation

Effect of EFV on Mo-MuLV Particle Production

<p>293 T cells were transfected with Mo-MuLV proviral DNA and treated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020119#ppat-0020119-g006" target="_blank">Figure 6</a>, except that cell lysates were immunoprecipitated with a Mo-MuLV CA antibody. All drugs were tested at 5 μM. Viral particle production was quantified as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020119#s4" target="_blank">Materials and Methods</a>. Data represent at least three independent experiments and are expressed as the mean ± standard error.</p

Effect of EFV on a Model Pol Construct in the Y2H System

<p>Yeast strain CTY10-5d expressing a model Pol construct as both bait and prey was assayed for β-gal activity in the presence of EFV. Results are expressed as fold increase in β-gal activity compared to the untreated control. Data represent at least three independent assays and are expressed as the mean ± standard error.</p

EFV Does Not Effect Virion Protein Processing

<p>Viral particles were pelleted from pDRNL-transfected 293T cells treated with 5 μM of each drug, and the Gag and Gag-Pol processing patterns were analyzed by quantitative Western blotting using (A) anti-CA and (C) anti-RT antibodies. Quantitation of the ratio of (B) p24 to Pr55^Gag and (D) p51 to p66 from Western blots. The ratios were calculated from at least three independent experiments and are expressed as the mean ± standard error.</p

Inhibition of Viral Particle Production by EFV Is Dependent on a Functional HIV-1 PR and Is Mediated by the Drug Binding to p66 RT

<p>293 T cells were transfected with (A) PR(-)pNL4.3 that contains an active site mutation in the HIV-1 PR or (B) K103NpNL4.3 that prevents EFV binding to RT. Cells were transfected and treated with Trans[³⁵S]-label as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020119#ppat-0020119-g006" target="_blank">Figure 6</a>. Top panels represent virion-associated Gag or p24 and bottom panels represent Gag or Gag processing products present in cell lysates. Quantitation of viral particle production in 293T cells transfected with (C) PR(-)pNL4.3 and (D) K103NpNL4.3. Data represent at least three independent experiments and are expressed as the mean ± standard error.</p

EFV, TMC120, and TMC125 Enhance Intracellular Gag and Gag-Pol Processing

<p>293T cells were transfected with pDRNL and treated with 5 μM of each drug. After 36 h post-transfection, cell lysates were harvested and viral proteins were detected by quantitative Western blot analysis using (A) anti-RT and (C) anti-CA antibodies. AlexFluor680 goat anti-mouse was used as the secondary antibody. Western blots were scanned on an Odyssey Infrared Imager and analyzed using Odyssey software. Quantitation of the intracellular ratio of (B) p51 to p66 and (D) p24 to Pr55^Gag. The ratios were calculated from at least three independent experiments and are expressed as the mean ± standard error.</p

NNRTI Enhancement of p66 Homodimer Formation In Vitro

<p>The effect of NNRTIs on the p66 monomer-homodimer equilibrium was determined by incubating His-p66 in the absence and presence of a 10-fold molar excess of drug relative to total protein for 2 h at 4 °C followed by analysis of the quaternary structure by SEC.</p

Potent NNRTIs Inhibit Viral Particle Production

<p>Viral particle production in drug-treated 293T cells (A) and in HeLa cells (C). Top panels represent virion-associated p24 and bottom panels are p24 present in cell lysates. Cells were transfected with pDRNL and treated with drug as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020119#ppat-0020119-g003" target="_blank">Figure 3</a>, except that after 36 h, cells were metabolically labeled with Trans[³⁵S]-label for 4 h. Cell lysates were immunoprecipitated using anti-CA mAb and viral particles in the supernatant were pelleted through a sucrose cushion. The proteins were separated by SDS-PAGE and visualized and quantified using a phosphorimager. Viral particle production in 293T cells (B) and HeLa cells (D) was quantified as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020119#s4" target="_blank">Materials and Methods</a>. Data represent at least three independent experiments and are expressed as the mean ± standard error.</p

Inhibition of vaginal transmission of HIV-1 in hu-SCID mice by the non-nucleoside reverse transcriptase inhibitor TMC120 in a gel formulation

OBJECTIVE: The development of drugs that can be used as topical microbicides is currently recognized as a priority area of research. DESIGN: A preclinical evaluation of the potential effectiveness of TMC120, a non-nucleoside reverse transcriptase inhibitor (NNRTI), as a topical microbicide to prevent vaginal HIV-1 transmission in a humanized severe combined immunodeficient (hu-SCID) mouse model. METHODS: Reconstituted mice received an intravaginal application of a TMC120-containing gel 20 min prior to a non-invasive vaginal challenge with cell-associated HIV. The possible cytotoxic effect of TMC120-containing-gel on lymphocytes was assessed and their in vivo migration was followed using fluorescently labelled human lymphocytes. Systemic infection was monitored by p24 antigen detection in culture supernatant from cocultured intraperitoneal cells using antigen capture enzyme-linked immunosorbent assay test and by the presence of integrated proviral HIV-1 DNA in DNA extracted from spleen cells. In vivo migration of labelled lymphocytes was examined by analysis of cells isolated from regional lymph nodes. RESULTS: In this model, systemic infection was successfully inhibited by the presence of TMC120-containing gel at vaginal level. The in vivo migration of human lymphocytes from the vagina to regional lymph nodes, following the deposition of TMC120-containing gel, excluded the possibility that inhibition of systemic infection was a result of NNRTI toxicity. CONCLUSIONS: Vaginal transmission of HIV was successfully prevented by the application of a gel formulation containing TMC120. This is the first evidence of the in vivo effectiveness of a microbicide preparation containing an NNRTI against cell-associated HIV.status: publishe