Formation of a Unique Cluster of G-Quadruplex Structures in the HIV-1 nef Coding Region: Implications for Antiviral Activity

G-quadruplexes are tetraplex structures of nucleic acids that can form in G-rich sequences. Their presence and functional role have been established in telomeres, oncogene promoters and coding regions of the human chromosome. In particular, they have been proposed to be directly involved in gene regulation at the level of transcription. Because the HIV-1 Nef protein is a fundamental factor for efficient viral replication, infectivity and pathogenesis in vitro and in vivo, we investigated G-quadruplex formation in the HIV-1 nef gene to assess the potential for viral inhibition through G-quadruplex stabilization. A comprehensive computational analysis of the nef coding region of available strains showed the presence of three conserved sequences that were uniquely clustered. Biophysical testing proved that G-quadruplex conformations were efficiently stabilized or induced by G-quadruplex ligands in all three sequences. Upon incubation with a G-quadruplex ligand, Nef expression was reduced in a reporter gene assay and Nef-dependent enhancement of HIV-1 infectivity was significantly repressed in an antiviral assay. These data constitute the first evidence of the possibility to regulate HIV-1 gene expression and infectivity through G-quadruplex targeting and therefore open a new avenue for viral treatment. © 2013 Perrone et al

Bimolecular Fluorescence Complementation Reveals that HIV-1 Nef Oligomerization is Essential for CD4 Downregulation and Viral Replication

HIV-1 Nef is a small myristoylated protein capable of interaction with a diverse array of host cell signaling molecules. Multi-faceted in its function, Nef is a critical accessory factor, essential for high-titer viral replication and AIDS progression. Despite its essential role, the molecular mechanisms of Nef-mediated HIV pathogenicity are not fully understood. Previous biochemical and structural studies have suggested that Nef may form homodimers and higher order oligomers in HIV-infected cells. The studies summarized below investigated the oligomeric status of the HIV-1 nef gene product and its role relative to Nef-mediated function. We explored the formation of Nef oligomers in live cells by adapting a bimolecular fluorescence complementation (BiFC) assay, a well-defined system in which dimeric protein interactions are observed in live cells. Using this assay, we provided the first direct evidence for Nef oligomerization in vivo. We then assessed the generality of oligomerization by a group of Nef alleles broadly representative of all major HIV-1 subtypes and found oligomerization was highly conserved across all subtypes examined. We then used our BiFC system to define residues previously suggested via X-ray crystallographic studies to comprise the Nef dimerization interface. Using a systematic strategy for the mutagenic profiling of the oligomerization interface, we discovered two classes of residues were critical to Nef oligomerization. BiFC was completely abolished when either all four key hydrophobic interactions were simultaneously removed or when ionic interactions mediated by D123 and R105 were disrupted. Finally, we utilized Nef mutants identified in the mutagenic profiling of the oligomerization interface to explore the effects of oligomeric disruption on Nef function. Screening a panel of Nef mutants with varying degrees of oligomeric disruption we discovered, surprisingly, despite the varying effects on oligomerization, all of these mutants were shown to dramatically disrupt Nef-induced CD4 downregulation and viral replication. Taken together, the studies presented in this dissertation advance the field of HIV research by furthering our understanding of the regulation of Nef-mediated downregulation of CD4 and enhancement of HIV replication as well as validating the Nef oligomerization interface as a potential target for anti-retroviral drug design

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-\u3baB 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

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

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

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

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