CD spectra of G-4 <i>nef</i> single-stranded or double-stranded oligonucleotides in the presence of G-4 ligands.

<p>A) CD spectra of G-4 <i>nef</i> single-stranded oligonucleotides in the presence of TMPyP4, BRACO-19 or PIPER. Addition of ligands stabilized G-4 conformations and generated ICD bands in the UV/Vis absorption regions of G-4 ligands. B) CD spectra of G-4 <i>nef</i> double-stranded oligonucleotides in the presence or absence of K⁺ and TMPyP4. Addition of the G-4 binding compound induced shifting from double-stranded DNA spectra to mixed type G-4 signatures in all three cases.</p

CD thermal unfolding of the G-4 <i>nef</i> oligonucleotides.

<p>CD spectra measured at increasing temperatures (25-95°C) are shown on the left. Arrows indicate spectral trends at the corresponding wavelengths. Asterisks indicate isosbestic points. Plots of molar ellipticity values (black circles) measured at the indicated wavelength (corresponding to positive peaks) as a function of temperature are reported on the right. Arrows indicate T_m points.</p

Taq polymerase stop assay.

<p>A) and B) Templates containing the G-4 nef sequences Nef8528, Nef8624 and Nef8547, a 4-T linker and a primer annealing region were allowed to fold and anneal to the P³²-5’-end labelled primer in K⁺ 100 mM, treated with increasing concentrations of TMPyP4 (0-2 µM) and subjected to Taq polymerase extension. The control template contained a sequence unable to fold in G-4, and the same 4-T linker and primer annealing region as the nef templates. A) The * symbol indicates pausing sites in the G-4 region of nef templates. The ¤ symbol indicates a polymerase stop site obtained prior to addition of TMPyP4 in Nef8624. Lanes 1, 7 and 13 (A), and lanes 1 and 6 (B) were Maxam and Gilbert marker lanes performed on the double stranded PCR amplified region. Markers indicate the C-rich complementary strand.</p

Putative G-forming regions in the HIV-1 <i>nef</i> coding region.

<p>A) Scheme of G-4 formation within the double-stranded DNA of the <i>nef</i> region: Nef8528, Nef8547, Nef8624 G-4 structures are shown in blue, green and red, respectively. The numbers of nts separating each G-4 structure are indicated. The scheme indicates the possibility of formation of a cluster of non-canonical DNA structures within a small portion (112 nts) of the HIV-1 genome. B) Nucleotide sequence of the <i>nef</i> coding region where three putative G-4 sequences were identified. Nef8528 is shown in blue and Nef8624 in red. Nef8547 was identified on the non-coding strand, thus the reverse complementary sequence is shown on the upper strand (in green). C) Scheme of the HIV-1 nef coding sequence with numbering referring to the HIV-1 strain HXB2/LAI, NC_001802. D) Scheme of the aminoacidic sequence of the Nef protein indicating reported structural domains [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073121#B73" target="_blank">73</a>]. The protein moiety coded by the G-4 rich nucleotide region is highlighted by the rectangular yellow shape, indicating involvement of the conserved N-terminal Nef core region. Note that the first three nts of the Nef8528 sequence exactly code for the first amino acid of the protein core region.</p

Effects of the stabilization of the <i>nef</i> G-4s by TMPyP4 on gene expression and viral infectivity in Nef sensitive cells.

<p>A) Effect of TMPyP4 and TMPyP2 on Nef-GFP expression measured by flow-cytometry. HEK 293T cells were transfected with a Nef-GFP encoding plasmid and treated with TMPyP4 or TMPyP2 (10 µM) for 24 h. Results are shown as percent mean of fluorescence relative to the control cells incubated ± SD (n = 4). Statistical difference was observed for TMPyP4 (p<0.05), but not for TMPyP2. B) and C) TZM-bl cells were infected with wild-type (black bars) and ΔNef (grey bars) HIV NL4-3 in the presence of either the G-4 ligand, TMPyP4 (B), or the negative control compound, TMPyP2 (C). After 48 h, infectivity was assessed as relative luciferase activity in infected cells. Results are shown as percent infectivity relative to the control cells incubated with carrier solvent (DMSO) ± SEM (n = 3). In B), no statistical difference was observed across ΔNef infected cells, even at the highest concentration (p > 0.15). In C), no statistical difference was observed for the wild-type virus at 3 µM and 6 µM (p >0.345 and >0.325, respectively) relative to the untreated control. The negative control compound, TMPyP2, further had no impact on the ΔNef virus at any concentration tested (p>0.29 at 6 µM). D) TZM-bl cell viability in the presence of compounds was assessed via the Cell-Titer Blue assay (Promega). TZM-bl cells were incubated with the indicated concentrations of compounds for 48 h and cell viability was assessed via the Cell-Titer Blue assay relative to control cells incubated with carrier solvent. Assays were done in triplicate.</p

A Dynamic G‑Quadruplex Region Regulates the HIV‑1 Long Terminal Repeat Promoter

G-Quadruplexes, noncanonical nucleic acid structures, act as silencers in the promoter regions of human genes; putative G-quadruplex forming sequences are also present in promoters of other mammals, yeasts, and prokaryotes. Here we show that also the HIV-1 LTR promoter exploits G-quadruplex-mediated transcriptional regulation with striking similarities to eukaryotic promoters and that treatment with a G-quadruplex ligand inhibits HIV-1 infectivity. Computational analysis on 953 HIV-1 strains substantiated a highly conserved G-rich sequence corresponding to Sp1 and NF-κB binding sites. Biophysical/biochemical analysis proved that two mutually exclusive parallel-like intramolecular G-quadruplexes, stabilized by small molecule ligands, primarily fold in this region. Mutations disrupting G-quadruplex formation enhanced HIV promoter activity in cells, whereas treatment with a G-quadruplex ligand impaired promoter activity and displayed antiviral effects. These findings disclose the possibility of inhibiting the HIV-1 LTR promoter by G-quadruplex-interacting small molecules, providing a new pathway to development of anti-HIV-1 drugs with unprecedented mechanism of action