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

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

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

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 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

Summary of resistance results obtained by all methodologies.^a

a<p>Plasma samples underwent testing by Sanger sequencing (SS), allele-specific PCR (AS-PCR) and ultra-deep sequencing (UDS) targeting mutations in HIV-1 reverse transcriptase.</p

Predictors of virologic suppression <400 copies/ml 4–12 months after restarting NNRTI-based ART.^a

a<p>The analysis included 90 patients who restarted NNRTI-based ART without a protease inhibitor and had at least one viral load measurement in the 4–12 months after re-starting therapy.</p>b<p>As noted above some patients had the viral load measured by assays with a lower limit of quantification of either 75 or 400 copies/ml. NNRTI = non-nucleoside reverse transcriptase inhibitor; ART = antiretroviral therapy; OR = Odds ratio; CI = confidence interval.</p

Characteristics of the study population that interrupted NNRTI-based ART in SMART, according to the interruption modality.^a

a<p>Patients interrupted ART by simultaneously interrupting all drugs, continuing the nucleos(t)ide reverse transcriptase inhibitors (NRTIs) for a short period, or switching to a ritonavir-boosted protease inhibitor for a short period, referred to as simultaneous, staggered and switched interruption respectively.</p>b<p>In 46 patients the viral load was measured by assays with a lower limit of quantification of either 75 or 400 copies/ml and results were “undetectable” below these cut-offs; 19 patients showed a viral load between 50 and 400 copies/ml. NNRTI = non-nucleoside reverse transcriptase inhibitor; ART = antiretroviral therapy; MSM = Men who have sex with men; IQR = interquartile range.</p