Effect of MAba4 on the HIV-1 IN activity.

<p>A: Standard concerted integration assays were performed with 1 pmole of IN in presence of increasing amounts of MAba4. The final NaCl concentration was adjusted to 30 mM. MAba4 was added to the mixture at different concentrations and the reaction products were loaded on a 1% agarose gel: 0 (lane 1), 10 (lane 2), 20 (lane 3), 30 (lane 4), 40 (lane 5), 50 (lane 6), 100 (lane 7), 200 lane (8), 600 (lane 9) or 800 ng (lane 10). The position and the structure of the different products obtained after half-site (HSI), full-site (FSI) and donor/donor integration (d/d) are indicated. B: Densitometry of the FSI (full site integration) and FSI+HIS (half site integration) bands of experiments shown in A. The different integration products were quantified using the Image J software. Panel values are the mean ± standard deviation (error bars) of three independent experiments. C: Inhibition assays were performed under different preincubation conditions. MAba4 was either added simultaneously to IN and DNA ([IN+DNA+MAba4]), either after preincubation between IN and DNA ([IN+DNA]+MAba4) or it was preincubated with IN before adding the DNA substrates ([IN+MAba4]+DNA). The different integration products detected on agarose gel were quantified using the Image J software. Panel values are the mean ± standard deviation (error bars) of three independent experiments.</p

Inhibition of binding of MAba4 to IN and K156 by DNA fragments.

<p>A: Histogram representation of competition ELISA results for the competitive binding between DNAs and MAba4 to IN and K156. Wells were coated either with IN and K156 or with the complexes IN-DNA and K156-DNA. IN or K156 where incubated either with DNA or added to the antibody- complex. From left to right: IN, IN-DNA complexes, K156, K156- DNA complexes. Panel values are the mean ± standard deviation (error bars) of three independent experiments. CRE (cAMP Responsive Element) was used as control. B: Histogram representation of simple ELISA results for the binding of DNAs to MAba4. Panel values are the mean ± standard deviation (error bars) of three independent experiments. C: Histogram representation of an ELISA control for the competitive binding of DNAs to IN and K156 realized in presence of a mouse IgA antibody.</p

Peptides and oligonucleotides used to prepare and study the epitope.

<p>A: The 29-mer peptide K159 (residues 147–175 in IN of HXB2) was used as immunizing peptide <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016001#pone.0016001-Sourgen1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016001#pone.0016001-Maroun1" target="_blank">[46]</a>. To delimit the epitope and analyze the properties we used several other peptides: pep-a4 reproducing the α4-helix sequence; IN636, a C-terminal fragment of K159 that has shown epitope properties in a previous work <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016001#pone.0016001-Maroun1" target="_blank">[46]</a>; IN638, a N-terminal fragment of K159; INH5, a strong inhibitor of IN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016001#pone.0016001-Maroun1" target="_blank">[46]</a>, that includes a loop region (residues 167–171) and the beginning of the α5-helix (residues 172–187); K156, a structural analogue of pep-a4, that is constrained into helix through seven helicogenic substitutions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016001#pone.0016001-Zargarian1" target="_blank">[26]</a>; HTH (α4-helix-loop-α5-helix) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016001#pone.0016001-Merad1" target="_blank">[27]</a>; E156, E159 and E148 (structural analogues of K156 in which residues Lys156, Lys159 and Gln148 has been respectively replaced by Glu); K156-E, the elongated K156 peptide ([136–146]-K156); and the peptide control that derives from K156 from six AA→Glu mutations. B: Once folded into hairpin structure around the central trinucleotide <b>TTT</b>, the three LTRoligonucleotides mimic the U5 LTR extremity. LTR17 carries a 7 pb stem corresponding to the most important region for IN binding. LTR34 carries a 17 pb stem, a minimum DNA size for successful reaction of integration <i>in vitro</i>. It represents an unprocessed version of U5 LTR, while LTR32 corresponds to the processed version with its 15 bp and its 5′CA hanging dipeptide. The DNA duplex CRE (cAMP responsive element) is used as a control.</p

Thermodynamic parameters for the binding of RAL to LTR32.

<p>Titration of LTR32, at three different concentrations: 9 nM (black), 20 nM (red), and 30 nM (blue). Curve treatment provided a 1∶1 stoichiometry for the complex formation and an average Kd of ≈6 nM for the binding affinity. Samples were in phosphate buffer pH 6, I = 0.05, at 5°C, MgCl₂ 5 mM final concentration.</p

Circular dichroism analysis of oligonucleotides-drug complexes.

<p>Spectra of LTR34 (A) and LTR32 (B) at 10 µM (black) and difference spectra [LTR32/34 (10 µM)+RAL (10 µM, red; 20 µM, green; 40 µM, blue; 60 µM, orange; and 80 µM, purple)−LTR32/34 (10 µM)], in phosphate buffer pH 6, I = 0.05, and MgCl₂ 5 mM final concentration.</p

MD simulations of the RAL-LTR34 and RAL-LTR32 complex systems (PFV oligonucleotides), using GROMACS with the AMBER force field.

<p>(A) Time evolution of RMSD (root mean square deviation) values based on all the heavy atoms for the two LTR34 trajectories (black: LTR34-1 and blue: LTR34-2). RMSD calculations for a single trajectory were also performed using the sugar C4′ atoms (green: LTR34-1) and repeated for LTR34 devoid of 3′-AT (purple). (B) Time evolution of RMSD values of LTR32 for two trajectories (black: LTR32-1 and blue: LTR32-2). (C) RMSF (root mean square fluctuation) variations of sugar C4′ atoms for LTR34 and (D) RMSF variations of sugar C4′ atoms for LTR32.</p

Calculated binding parameters for the complexes of RAL with LTR32 and LTR34.

<p>The free energy ΔG_MMPBSA from two trajectories for each system (LTR34-1, 2 and LTR32-1, 2) and averaged over 500 frames from each trajectory. Energies and standard deviations are given in kcal/mol. E_ele: Coulombic energy; E_vdw: van der Waals energy; E_MM: total molecular mechanics energy (E_ele+E_vdw); G_PB: polar solvation free energy based on Poisson-Boltzmann; G_SASA: Non-polar solvations free energy based on SASA; TΔS: the entropy contribution to the binding calculated by the QH; ΔG: the total free energy.</p

Effects of RAL on the distance and the fraying of unprocessed LTR ends.

<p>Time evolution of the distance (nm) between the mass of RAL (D) and that of the terminal bases (T and A) of LTR34-1 (black:D-T and red: D-A) and LTR34-2 (purple: D-T and blue: D-A). Interaction of RAL with the terminal bases decreases the distance between the drug and the bases and also reduces moderately the end fraying.</p

UV-absorption analysis of oligonucleotides.

<p>Spectra of RAL 20 µM (black) together with 20 µM LTR32 (green), 20 µM LTR34 (blue), 20 µM LTR30 (red), in phosphate buffer pH 6, I = 0.05, and MgCl₂ 5 mM final concentration.</p

Snapshots from the two 100 ns trajectories of RAL in complex with unprocessed LTR (LTR34-1 and 2, top) and processed LTR (LTR32-1 and 2, bottom).

<p>RAL is colored in slime green and bases in sandy brown, except for atoms at interacting distances which are colored using the usual code (hydrogen in white; nitrogen in blue; and oxygen in red) except for carbons, while the Mg²⁺ ion is represented by magenta ball. Selected snapshots are 0 ns (the initial structure), 50 ns and 100 ns.</p