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

Abstract

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-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.

Visualization of DNA G-quadruplexes in herpes simplex virus 1-infected cells

“…Three HSV-1 sequences were tested: un2 (antiparallel), un3 (parallel) and gp054a (mixed-type); un1 could not be tested because unstable in solution (17). A series of control sequences was alongside tested: the tetramolecular Oxytrichia telomeric G4, which 1H6 was developed against (Oxy2), the monomolecular Oxytrichia G4 (OxyTel), three different-length human telomeric G4s (hTel21, hTel22 and hTel54), four G4s of oncogene promoters (bcl-2, c-myc, c-kit1, c-kit2) (24–27), two G-rich oligonucleotides corresponding to the scrambled sequences of the human telomeric and HIV-1 LTR promoter G4s (hTelScra and LTRScra) (13,15,17), one random ssDNA (rnd), two polyTs (T9 and T30), one hairpin oligonucleotide (hp) and a dsDNA. RNA sequences were also tested, in particular: two cellular G4s found in the 5′-UTR of the transcript of the human NRAS proto-oncogene (NRAS) (28) and in telomeric transcripts (hTel 2 ) (29), two G4s that form in the HIV-1 RNA genome (U3-III and U3-IV) (30); one hairpin RNA from the HIV transcript (TAR) (31) and one unstructured random RNA (rndR).…”

“…Both in the case of HSV-1 and HIV-1, treatment of infected cells with G4 ligands greatly impaired viral infectivity (13,15,17); in particular, treatment with BRACO-19 stabilized G4s in the HSV-1 genome and inhibited viral replication (17). …”

Deficiency in DNA damage response, a new characteristic of cells infected with latent HIV-1

“…We initially focused on G4 binding agents that stabilize G-quadruplexes, since they were demonstrated to have activity toward G-rich RNA and DNA sequences of HIV-1. [25][26][27] In anticancer research, G4 binding agents were shown to trigger formation of DSBs and to induce synthetic lethality when combined with DNA repair inhibitors. 31,32 We hypothesized that similar effects might be induced in HIV-1 latent reservoirs by targeting HIV-1 provirus incorporated into the host chromosome.…”

“…[23][24][25][26][27][28] The cellular effects of G4 binding agents have been intensively investigated in anticancer research. 29,30 Studies showed that G4-stabilizing agents can induce DSBs, and sensitize cells to inhibitors of DNA repair, triggering apoptosis.…”

A Matter of Location: Influence of G-Quadruplexes on Escherichia coli Gene Expression

“…Recent studies investigated the influence of transcription-induced quadruplex formation in a double-stranded DNA (dsDNA) template. Quadruplexes can be induced far away from a transcription start site (TSS), functioning as silencer or enhancer of transcription (Perrone et al, 2013;Zhang et al, 2013). In addition to 5 0 untranslated regions (UTRs), potential quadruplexes have also been identified in the 3 0 -UTRs near transcription termination, splicing, and polyadenylation sites Beaudoin and Perreault, 2013).…”