A dynamic G-quadruplex region regulates the HIV-1 long terminal repeat promoter
G-quadruplexes, non-canonical 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-\xce\xbaB 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.
Introduction<!>Putative G-quadruplex sequences are present in the NF-\xce\xbaB and Sp1 binding sites of the HIV-1 LTR promoter region<!>Two stable G-quadruplexes form in the HIV-1 LTR<!>Multiple G-quadruplexes form in the full-length LTR G-rich region<!>Melting experiments<!>Footprinting studies<!>Taq polymerase stop assays<!>A novel drug-induced LTR structure<!>G-quadruplex structures regulate HIV-1 LTR promoter activity<!>The G-quadruplex ligand, BRACO-19, displays antiviral activity<!>Discussion<!>Materials<!>G-quadruplex analysis of the HIV-1 LTR region<!>Spectroscopic analysis<!>Taq polymerase stop assay<!>Footprinting assays<!>Plasmid construction<!>Reporter assays<!>Antiviral assays<!>Cytotoxicity Assays<!>
<p>G-rich sequences in DNA may associate to form four-guanine square planar structures called G-quartets which give rise to G-quadruplexes by stacking on top of each other. G-quadruplexes are polymorphic intra- or inter-molecular four-stranded structures, and the adopted structures depend on base sequence, strand concentration, loop connectivities, and cations present. DNA strands in G-quadruplex take anti-parallel, parallel, or hybrid conformations, and the nucleotide linkers between G-quartets can adopt a multitude of loop structures1.</p><p>G-quadruplexes are involved in several key biological functions2. In particular, tight clusters of putative G-quadruplex-forming sequences are located immediately upstream and downstream of the transcription start site in proliferation-associated genes3, 4. Notably, G-quadruplexes are found in the promoters of a wide range of genes important in cell signaling, having representatives from the six hallmarks of cancer5. Strong evidence of G-quadruplex formation inside human cells has been recently provided by an antibody-based approach2; moreover, G-quadruplex stabilizing and destabilizing proteins have been identified6, 7.</p><p>The biological relevance of G-quadruplex structures prompted the development of a diverse array of G-quadruplex ligands. Their general features comprise a large flat aromatic surface and cationic groups8: examples include perylenes (PIPER), porphyrins, (TMPyP4), and trisubstituted acridines (BRACO-19).</p><p>Besides humans, other mammals9, yeasts10 and prokaryotic cells11–13 also exhibit putative G-quadruplex forming sequences in regions proximal to the transcription start sites of protein-coding genes. Other organisms, such as viruses, might have evolved analogous regulatory mechanisms, but research in this area has not been explored.</p><p>Few data are available on the presence of G-quadruplexes in HIV-1: it has been demonstrated that a single-stranded portion (central DNA flap) of the reverse-transcribed pre-integration HIV-1 genome forms a G-quadruplex structure that specifically interacts with the viral nucleocapsid protein thereby protecting the pre-integrated genome from nuclease degradation14. Furthermore, a G-quadruplex structure apparently promotes dimerization of the two viral RNA genome copies15. In the proviral genome, however, no G-quadruplex structures have been reported.</p><p>Within the integrated provirus, the HIV-1 long terminal repeat (LTR) governs transcription of viral genes and the full-length genome16, 17. Two identical LTR sequences are located at both extremes of the HIV-1 provirus, each composed of three regions: U3, R and U5. The U3 promoter region can be divided in three functional sections: an upstream regulatory element (−454 to −104) including binding sites for cellular transcription factors, an enhancer (−105 to −79) with two binding sites for the nuclear factor κB (NF-κB), and the core promoter (−78 to −1) composed of three tandem binding sites for specificity protein 1 (Sp1) and a TATA box. The R (+1 to +96) region contains the unique transactivation response enhancer TAR (+1 to +60), which modulates Tat activation18.</p><p>Transcription of the HIV-1 provirus starts when the U3 promoter sequence binds cellular factors, including NF-κB, Sp1, the TATA box binding protein, and RNA polymerase II16. The transcription complex allows low-level production of viral transcripts. Interaction of the viral protein Tat with the 5'-end of nascent viral transcripts greatly amplifies gene expression19.</p><p>Genetic variability within LTR binding sites in U3 and TAR regions has been observed in several HIV-1 subtypes20, 21. However, NF-κB and Sp1 binding sites are remarkably conserved22.</p><p>Here we show the first evidence of dynamic regulatory G-quadruplexes in the HIV-1 LTR region. Specifically, the conserved region encompassing NF-κB site and Sp1 sites contains G-tracts involved in two mutually exclusive G-quadruplex structures. G-quadruplex ligands greatly stabilized these conformations and induced folding and stabilization of an additional quadruplex. Mutations abolishing G-quadruplex formation enhanced LTR promoter activity, while G-quadruplex ligands depleted promoter activity and exerted antiviral activity, indicating a regulatory role for LTR G-quadruplexes and their possible exploitation as therapeutic targets.</p><!><p>The U3 region of the HIV-1 LTR (representative strain HXB2_LAI; NC_001802) is very rich in G bases: the segment corresponding to part of the core promoter and enhancer (positions −105/−48 with respect to the transcription initiation site) consists of 50% G and 70% GC. Gs were mainly clustered in groups of 2–4 continuous G bases (Fig. 1A). Because of these features, the possibility of G-quadruplex folding was analyzed using an online algorithm-based software for recognition of putative Quadruplex forming G-Rich Sequences (QGRS Mapper)23. Putative QGRS were ranked based on G-score, the likelihood to form a stable G-quadruplex. Twelve putative sequences were found (Table S1): 3 sequences, namely LTR-I, -II and -III, were composed of GGG repeats (numbered 1–6, Fig. 1A) and could thus generate G-quadruplexes with 3 stacked G-quartets exhibiting the highest G-scores. These sequences differed by the length of the loop segments connecting G-tracts (Table S1). The remaining 9 sequences involved 2 stacked quartets and were ranked with lower G scores (Fig. S1A).</p><p>Interestingly, the G-rich −105/−48 tract was also the binding region of two important cellular transcription factors that stimulate viral transcription: NF-κB, with two binding sites at −105/−96 and −92/−82, and Sp1, three molecules of which bind at −79/−68, −67/−57 and −56/−4817 (Fig. 1A). To establish the importance of these predicted sequences within a viral context, the degree of base conservation among HIV-1 strains was assessed. Among 953 analyzed LTR sequences, most Gs and also non-G bases were highly conserved (Fig. S1B).</p><!><p>The three LTR sequences, namely LTR-I, -II and –III, embedding four GGG tracts each (Fig. 1A) and displaying higher G-scores (Table S1), were selected and analyzed for their ability to form G-quadruplexes. Circular dichroism (CD) spectroscopy was initially performed in the absence or presence of increasing concentrations of K+ to monitor G-quadruplex formation and its likely topology. LTR-II and LTR-III produced CD spectra characteristic of a G-quadruplex structure in a K+-dependent manner (Fig. 1B, Fig. S2A). Spectra of both oligonucleotides exhibited a positive band at 266 nm and a negative peak at 244 nm, a signature suggesting a parallel-like G-quadruplex24, 25. In contrast, LTR-I showed a negative band at 240 nm, a shoulder at 260 nm and a positive peak at 280 nm, and low K+-dependence, features not typical of G-quadruplex structures (Fig. S2A). The topology of the selected sequences was further assessed by UV thermal difference spectroscopy (TDS). LTR-II and LTR-III produced TDS signatures with three positive bands at 275, 257 and 239 nm, and three negative bands at 296, 261 and 252 nm, characteristic of the G-quadruplex structure26 (Fig. 1C). In addition, the TSD factor, calculated as ΔA240nm/ΔA295nm was above 5, indicating a parallel-like topology (Fig. 1C; inset)24. In contrast, the TDS signature of LTR-I was not representative of G-quadruplex (Fig. S2B).</p><p>Further support for the formation of G-quadruplexes was obtained by protection assays. Two G-N7 alkylators were separately employed: DMS, the standard reactant to highlight G bases involved in G-quartets, and clerocidin (CL), endowed with finely tunable reactivity27, 28. In the CL protection assay, tracts 2, 3, 4 and 5 in the LTR-II sequence were all protected (Fig. 1D). In particular, in G-tract 2, the three 5'-Gs were involved in G-quadruplex, while the 3'-G was excluded (symbol *, lane 10). Surprisingly, in tract 3 the 5'-G was overexposed to cleavage (symbol ¤, lane 10). Based on CL discrimination between protected and stretched bases28, the cleaved G, adjacent to the 8-nt-long linker region, is likely strained and therefore exposed to CL alkylation. In the LTR-III sequence, tracts 3, 4, 5, and 6 were clearly protected, while the 3'-G of tract 6 was not (symbol *, lane 15). Conversely, no protection was observed in the LTR-I sequence (lanes 1–5). Similar results were obtained using the G-N7 alkylator DMS (Fig. S3).</p><p>Stability of LTR-II and LTR-III G-quadruplexes in the absence/presence of 100 mM K+ was next assessed by melting experiments monitored by CD (Table 1 and Fig. S4A). In the presence of K+, LTR-III was 4°C more stable than LTR-II; Tm values of LTR-II and LTR-III unfolding were 2.6°C and 1.8°C (respectively) higher than Tm values gained during the refolding process (Fig. S4B). The small hysteresis was indicative of reversible and intramolecular G-quadruplex formation, with folding kinetics faster for LTR-III than for LTR-II. These data indicate that the presence of two long loops in LTR-II (9, 11, 1 nt-loops) versus only one long loop in LTR-III (11, 1, 3 nt-loops) moderately affected both the thermodynamic stability and the kinetics of G-quadruplex formation. UV melting experiments at 4 µM and 60 µM oligomer concentrations resulted in superimposable Tm values, confirming intramolecular G-quadruplex formation (Fig. S4C).</p><p>The commercially available G-quadruplex ligands TMPyP4, BRACO-19 and PIPER were next tested by CD for binding to the novel LTR G-quadruplex structures in the absence/presence of 100 mM K+. In the presence of K+, the average ΔTm was above 30°C indicating both LTR sequences were significantly stabilized by the compounds. BRACO-19 was most efficient, followed by TMPyP4; PIPER was less effective, especially with LTR-III. Interestingly, even in the absence of K+, compounds induced a G-quadruplex conformation with average ΔTm around 15°C (Table 1). Further evidence of the stability of the LTR G–quadruplexes was provided by Taq polymerase stop assay (Fig. 1E). Samples were incubated in the absence/presence of 100 mM K+ (lanes 1 and 2), and with increasing concentrations (50–100 nM) of TMPyP4 (lanes 3 and 4). TMPyP2, a non-G-quadruplex binding porphyrin29, was used as a negative control (lanes 5–6). Taq polymerase activity was tested at 47°C against LTR-I, LTR-II and LTR-III DNA templates. Full-length products were obtained in the absence of K+ in the three sequences. However, in the presence of K+, a premature stop site occurred in LTR-II and LTR-III (lanes 2) at the first two 3'-G bases involved in G-quadruplex (G-tract 5 in LTR-II and 6 in LTR-III). The stop became clearer upon incubation with TMPyP4 (lanes 3–4). The stop observed in the presence of TMPyP2 was comparable to the stop with K+ alone (compare lanes 5–6 with 2), confirming that the compound did not affect G-quadruplex formation. In addition, no stop was observed in LTR-I, showing that polymerase activity was not directly inhibited by the compounds (LTR-I, lanes 2–6).</p><!><p>The full-length LTR G-rich sequence comprises 4 GGG-tracts (1, 3–5), 2 GGGG-tracts (2, 6) and 3 additional GG-tracts (3', 3", 6') which could be used for multiple G-quadruplex formation (Fig. 1A and Fig. S1A).</p><p>We examined a number of sub-sequences to dissect this issue, corresponding to LTR-II, LTR-III and their combination of LTR-(II+III).</p><!><p>CD-monitored melting experiments (Table 1) showed that the melting temperatures of LTR-II and LTR-III increased by 10–13°C in the presence of K+. LTR-(II+III) and the full length LTR (FL-LTR) were also greatly stabilized by the metal ion, Tms being very close to each other and 4–6°C higher than for LTR-II and LTR-III. G-quadruplex ligands further stabilized these conformations (Table 1).</p><!><p>LTR-(II+III) and FL-LTR were next examined in a CL-protection assay (Fig. 2). In the LTR-(II+III) sequence, all G-tracts present (2–6) were protected indicating the coexistence of LTR-II and LTR-III structures (compare lanes 5 and 4, Fig. 2A). In the FL-LTR, G-tract 1 was not protected (compare lanes 4 and 3, Fig. 2B), showing its exclusion from G-quadruplex folding. In the 3'-region of FL-LTR, magnified in Fig. 2C, G-tracts 4, 5 and 6 were clearly protected (compare lanes 4 and 3, Fig. 2C). Tracts 3 and 2 were partially protected (G −77, G −90 and G −87) and partially overexposed (G −76, G −78 and G −89) (symbols * and ¤, respectively, lane 4, Fig. 2C), indicating involvement of these tracts in the G-quadruplex, with buried and stretched bases. In addition, G-71 was protected suggesting participation in the G-quadruplex conformation or burial within the long linker region possibly folded on the quadruplex core. These data imply that both LTR-II and LTR-III conformations form in the FL-LTR. To note that similar dynamic G-quadruplexes have been previously proposed30. In addition, both LTR-II and LTR-III display a 11-bp long loop that could form 4 Watson-Crick base pairs and a GTG hairpin. Similar folding in the loop of the hTERT promoter G-quadruplex structure has been shown to promote cooperative binding31, 32. However, based on the footprinting data, such a folding does not likely occur in the LTR G-quadruplexes.</p><!><p>In Taq polymerase stop assays, at increasing concentrations of K+, pausing sites were observed at all G-quadruplex relevant G-tracts, i.e. 6, 5, 4, 3 and 2 (lanes 2–4, Fig. 3A), confirming the coexistence of LTR-II and LTR-III structures. With TMPyP4, two major stops emerged at G-tracts 6 and 5, while pausing at G-tracts 4–2 was no longer observed (lanes 5–6). Using BRACO-19, an identical behavior was obtained (lanes 9–10), whereas the control compound TMPyP2 maintained a stop site pattern similar to that observed in K+ alone (lanes 7–8). The strong stabilization imparted by the ligands (see Table 1) apparently caused the polymerase to stop at the first G repeat involved in a G-quadruplex, without allowing the enzyme to further proceed on the DNA template. We concluded that BRACO-19 and TMPyP4 mainly stimulated formation of an LTR-III-like structure and in part of an LTR-II-like structure (3'-stops at 6 and 5, respectively, Fig. 3A).</p><!><p>Interestingly, a new pausing site arose at G-tract 6', which suggests the formation a new LTR structure (LTR-IV).</p><p>Single-base mutations were next introduced in the FL-LTR to assess the role of each G-tract on G-quadruplex formation. Mutants were evaluated in the absence/presence of BRACO-19 and TMPyP4. In the presence of BRACO-19, deletion of one G base in G-tract 6 (m6) resulted in maintenance of the third stop site only at G-tracts 5/4 (formation of only LTR-II, Fig. 3B). Mutations in G-tracts 5 and 4 (m5 and m4) produced the harshest effects with all stops removed (no LTR G-quadruplex formed). Mutation in G-tract 3 (m3) increased the efficiency of the newly proposed first stop site (LTR-IV), while removal of one G in G-tract 6' (m6') effectively deleted the first stop (only LTR-II and LTR-III formed). Mutation in G-tract 2 (m2) hampered formation of the third stop (no LTR-II), while mutations in G-tracts 1 (m1), 3' (m3') and 3" (m3") produced no effect. Similar results were obtained with TMPyP4 (Fig. S5).</p><p>Deletion of G-tracts m6', m6, m5 and m4 inhibited formation of LTR-IV, indicating that these G-tracts must all be involved in its structure. The 19-bp LTR-IV sequence including these G-tracts revealed CD and UV features characteristic of a parallel G-quadruplex (Fig. S6), showing increased stability upon K+ addition (Table 1). Although in the presence of 100 mM K+ LTR-IV was less stable than LTR-II, LTR-III, LTR-(II+III) and LTR-FL, upon addition of G-quadruplex ligands, LTR-IV stability significantly increased (Table 1); in particular, Tm values of LTR-IV in the presence of ligands were consistently higher than Tm of LTR-III and LTR-II (Table 1). These data corroborate the results presented in Fig. 3A and 3B and demonstrate that G-quadruplex ligands both stabilize naturally-occurring G-quadruplex conformations and induce (and greatly stabilize) a novel G-quadruplex structure which is not present under physiological conditions. Interestingly, LTR-IV could form a G-quadruplex with a single bulge, a type of structure recently reported in G-quadruplex conformations33.</p><p>Based on these data, involvement of G-tracts and G-quadruplex types could be extrapolated as shown in Fig. 3C and Fig. S7. In the WT sequence, in the presence of ligands, three G-quadruplexes corresponding to LTR-II, LTR-III and LTR-IV are present. The core of the LTR G-quadruplex architecture rests on G-tracts 4 and 5 whose mutations completely abolish quadruplex building.</p><!><p>To evaluate the biological significance of G-quadruplex structures within the LTR sequence, the WT LTR sequence (nts −381/+83) and selected point mutants were cloned upstream of the firefly luciferase gene in a promotorless plasmid. A control vector with Renilla luciferase under the HSV-1 TK promoter was used to normalize transfection efficiency. Mutants corresponding to m4 and m5, which totally prevent G-quadruplex formation as described above, were assayed along with WT FL-LTR and mutant m3" which served as controls, respectively, for the original G-quadruplex-forming sequence and for a mutated sequence that does not disrupt G-quadruplex. LTR promoter activity was tested in HEK 293T cells: m4 and m5 LTR promoter activities were about twice as high as that of the WT LTR (Fig. 4A). This increment is in line with that observed in eukaryotic promoters, i.e. human c-Myc, KRAS and thymidine kinase 134–36. In contrast, when G-quadruplex formation was unharmed (m3"), LTR promoter activity was comparable to wild-type. These data suggest that G-quadruplexes act as repressor elements in the transcriptional activation of HIV-1.</p><p>The promoter activities of WT and m5 LTRs were next tested in the presence of increasing concentrations of BRACO-19. As shown in Fig. 4B, WT LTR promoter activity decreased to ~70% of the untreated control, while displaying no effect on m5 LTR activity, supporting a G-quadruplex-mediated inhibition. We also observed concentration-dependent inhibition of the control Renilla luciferase reporter gene by BRACO-19 (less intense than inhibition of the LTR-driven firefly luciferase) attributable to the toxic effects produced by ligand binding to eukaryotic G-quadruplex structures (data not shown). A similar effect has been previously observed34. Therefore, the effects of G-quadruplex ligands were next evaluated in a GFP-reporter system in the presence/absence of the transcriptional activator Tat, using flow cytometry to gate only GFP+ cells with viable morphology. In the presence of Tat, BRACO-19 was able to impair LTR promoter activity in a dose-dependent fashion (~50% inhibition at 6 µM, Fig. 4C). Moreover, in the absence of Tat, inhibition was 30%, confirming the results obtained in the luciferase assay. In contrast, the control ligand TMPyP2 did not show significant inhibition under these conditions (Fig. S8). Because Tat-mediated trans-activation boosts viral transcription acting on early mRNA transcripts originating by LTR-mediated transcription, when the latter is blocked, a magnified inhibition may be observed in the presence of Tat.</p><!><p>Having observed a reduction in HIV LTR-driven promoter activity, we next assayed the most active compound, BRACO-19, for antiviral activity. For these experiments, we used the TZM-bl assay, in which HIV infection drives transcription of an HIV-1 LTR-luciferase reporter gene construct37. We observed a significant inhibition of HIV-dependent gene expression upon treatment with the G-quadruplex ligand (Fig. 4D) at ligand concentrations that did not affect cell viability (Fig. S8C). HIV LTR activity in TZM-bl cells has been previously reported to be sensitive to the HIV accessory protein Nef38. To rule out a role for Nef in BRACO-19-mediated inhibition, the same experiment was repeated with Nef-defective HIV (MNef). A similar inhibitory effect was observed indicating that the effect of BRACO-19 is Nef-independent. In contrast, the control ligand TMPyP2 did not affect LTR activity in response to either virus (Fig. S8B). These data indicate that G-quadruplex ligands significantly inhibit HIV-1 and that this effect may depend on stabilization of G-quadruplex structures within the HIV-1 LTR promoter region.</p><!><p>We have presented the first evidence of a regulatory G-quadruplex-forming region in the HIV-1 LTR promoter. All main G-quadruplexes identified, i.e. LTR-III, LTR-II and LTR-IV, displayed sequence and structural features typical of G-quadruplexes previously implicated in regulation of the promoter activity of eukaryotic oncogenes, i.e. parallel-like topology, G3N1G3 motif (i.e. G3CG3)39, and silencing effect on promoter activity35, 36, 40–42.</p><p>Notably, a lentiviral plasmid engineered with point mutations in the Sp1 binding sites, mimicking our LTR-III-disrupting mutants (i.e. m4 or m6), reported reduced occupancy by HDAC1 resulting in 2–3 fold increased rates of spontaneous transcription activation compared to the WT-provirus17. It is therefore conceivable that HDAC1 participates in stabilization of LTR G-quadruplexes to down-regulate viral transcription.</p><p>The above described features point out the biological relevance of the G-quadruplex forming region in the HIV-1 promoter. This novel notion opens up the possibility of inhibiting the HIV-1 LTR promoter activity by G-quadruplex interacting small molecules. Presently, a major disadvantage of existing G-quadruplex ligands is that they recognize the quadruplex core without being able to finely discriminate between G-quadruplex structures. Here we have nonetheless shown that BRACO-19 is effective towards the virus with minimal effect on cell viability. Therefore a therapeutic window can be envisaged that would consent the employment of known G-4 binders as anti-HIV compounds. In addition, based on the sequence and structural differences between the LTR and eukaryotic G-quadruplexes, rational design of small-molecules may be envisaged for the development of anti-HIV-1 drugs with an unprecedented mechanism of action.</p><!><p>Oligonucleotides were from Sigma-Aldrich (Milan, Italy) (Table S1). Clerocidin (CL) was a gift of Leo Pharmaceutical Products (Ballerup, Denmark). Dimethylsulfate (DMS) was from Sigma Aldrich, TMPyP4 and PIPER from Calbiochem, (Merck Chemicals, Nottingham, UK), BRACO-19 from ENDOTHERM, (Saarbruecken, Germany). T4 polynucleotide kinase was from Invitrogen (Paisley, UK), [γ-32P]ATP from Perkin Elmer (MA, USA).</p><!><p>The HIV-1 LTR region (strain HXB2_LAI; NC_001802) was analyzed by QGRS Mapper (http://bioinformatics.ramapo.edu/QGRS/index.php) for prediction of G-4 forming sequences in both coding and non-coding strands. The following restrictions were applied: maximum length 30 nt; minimum G-group size 2 nt; loop size 0–15 nt. Conserved bases were evaluated by aligning LTR U3 sequences from the HIV database (http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html) using Jalview (http://www.jalview.org/).</p><!><p>For CD analysis, all DNA oligonucleotides were diluted from stock to final concentration (4 µM) in lithium cacodylate buffer (10 mM, pH 7.4) and, where appropriate, KCl. All samples were annealed by heating at 95 °C for 5 min, gradually cooled to room temperature and measured after 24 h. Compounds at 16 µM final concentration were added after DNA annealing. CD spectra were recorded on a Jasco-810 spectropolarimeter (Jasco, Easton, MD, USA) equipped with a Peltier temperature controller using a quartz cell of 5-mm optical path length and an instrument scanning speed of 100 nm/min with a response time of 4s over a wavelength range of 230–320 nm. The reported spectrum of each sample represents the average of 2 scans at 20°C and is baseline-corrected for signal contributions due to the buffer. Observed ellipticities were converted to mean residue ellipticity (θ) = deg × cm2 × dmol−1 (mol. ellip.). For the determination of Tm, spectra were recorded over a temperature range of 20–95 °C, with temperature increase of 5 °C/min or 2°C/min, followed by an equilibration step of 1 min. As previously described, Tm values were calculated according to the van't Hoff equation, applied for a two state transition from a folded to unfolded state, assuming that the heat capacity of the folded and unfolded states are equal43, 44. For thermal difference spectrum (TDS) analysis, UV spectra were recorded on Lamba25 UV/Vis spectrometer (Perkin Elmer) equipped with a Peltier temperature controller. TDS spectra were calculated by subtracting the spectrum at each temperature from the spectrum at 95°C. TDS factors were calculated as the absolute values of ΔA240nm/ΔA295nm, where ΔAλ is the difference, at the given wavelength λ, between the absorbance above and below the melting.</p><!><p>Primer (LTR G4 Taq primer, Table S1) was 5'-end labelled with [γ-32P]ATP using T4 polynucleotide kinase at 37°C for 30 min. The labelled primer (72 nM), annealed to the template (36 nM) in lithium cacodylate buffer (10 mM, pH 7.4), was extended with AmpliTaq Gold DNA polymerase (2U/reaction, Applied Biosystem, California) at 47°C for 30 min. Where specified, samples were incubated with G-quadruplex-ligands and 100 mM KCl for 20 min at room temperature and primer extension performed as described. Reactions were stopped by EtOH precipitation, extension products were separated on 12% denaturing gel, visualized by phosphorimaging (Typhoon FLA9000, GE Healthcare).</p><!><p>Clerocidin and DMS-mediated footprinting assays were performed as previously described28.</p><!><p>The LTR DNA region was amplified by PCR on the HIV-1 genome (AF033819.3) using reported primers (Table S1). The LTR amplicon was subcloned into pGL4.10-Luc2 (Promega) within XhoI and HindIII sites. The resulting pGL4.10-Luc2/LTR vector contained the sequenced 464 bp-long LTR-region (corresponding to nts −381/+83 in the HIV-1 genome) fused to the luciferase coding region. Mutant pGL4.10-Luc2/LTR vectors were generated using QuikChange mutagenesis kit (Stratagene/Agilent Technologies) and primers (Table S1).</p><!><p>Vectors pGL4.10Luc2/LTR and pGL4.74 (200 ng each) were transfected in 5.5×104 HEK 293T cells per well onto 96-well plates, using TransIT-293 Transfection Reagent (Mirus Bio LLC). Plasmid pGL4.74-hRLUC/TK (Promega), containing a Renilla luciferase gene driven by the Herpes Simplex virus thymidine kinase promoter, was used as a control for transfection efficiency. Expression of firefly luciferase, with respect to that of Renilla luciferase, was determined 24 h after transfection using Dual-Glo Luciferase Assay System (Promega). Cell lysate (75 µL) was mixed with reconstituted Dual-Glo Luciferase Buffer (75 µL) or Dual-Glo Stop&Glo Buffer, and light output detected with VICTOR X2 Multilabel Plate Reader (Perkin Elmer). Luciferase and Renilla output ratio was calculated.</p><p>For the LTR-GFP reporter assay, 2×105 of HEK 293T cells in 6-well plates were transfected with pcLTR-EGFP DNA 3.1. After 4 h, cells were treated with BRACO-19 or TMPyP2 and incubated over-night. To evaluate mean of EGFP fluorescence, a total of 30000 events were acquired for each sample with an LRS 2 instrument using FACS DIVA Software (BD Bioscience) and analyzed with Flow Jo (Tree Star).</p><!><p>Viral stocks were prepared by transfection of HEK 293T cells with wild-type and Nef-defective (ΔNef) proviral genomes (NL4-3 strain) and amplified in the T cell line, MT2 (NIH AIDS Research and Reference Reagent Program) as previously described45, 46. Viral transcription was measured using the TZM-bl reporter cell line which contains a luciferase reporter under the control of the HIV-1 LTR37 (NIH AIDS Research and Reference Reagent Program). Cells were seeded in 96-well plates (2×104) and grown overnight. BRACO-19 and TMPypP2 were preincubated separately with both cell culture medium (100 µL) and wild-type HIV-1 or ΔNef HIV-1 (100 µL) for 4 h prior to infection in a combined final volume of 200 µL. After 48 h at 37°C, cells were washed with PBS and lysed in luciferase lysis buffer (Promega). Lysates (40 µL) with 50 µL luciferase reagent (Promega) were read with a delay time of 2 s and an integration period of 10 s.</p><!><p>TZM-bl cells37 were seeded in 96-well plates (2×104), grown overnight incubated with compounds in DMSO carrier solvent and incubated at 37°C. After 48 h, cytotoxicity was assessed using the Cell Titer Blue reagent (Promega).</p><!><p>Supporting Information Available: Supporting Figures S1–S8 and Tables S1–S2. This material is available free of charge via the Internet at http://pubs.acs.org.</p>