G-quadruplexes (G4s) are non-canonical nucleic acids secondary structures that form within guanine-rich strands of regulatory genomic regions. G4s have been extensively described in the human genome, especially in telomeres and oncogene promoters; in recent years the presence of G4s in viruses has attracted increasing interest. Indeed, G4s have been reported in several viruses, including those involved in recent epidemics, such as the Zika and Ebola viruses. Viral G4s are usually located in regulatory regions of the genome and implicated in the control of key viral processes; in some cases, they have been involved also in viral latency. In this context, G4 ligands have been developed and tested both as tools to study the complexity of G4-mediated mechanisms in the viral life cycle, and as therapeutic agents. In general, G4 ligands showed promising antiviral activity, with G4-mediated mechanisms of action both at the genome and transcript level. This review aims to provide an updated close-up of the literature on G4s in viruses. The current state of the art of G4 ligands in antiviral research is also reported, with particular focus on the structural and physicochemical requirements for optimal biological activity. The achievements and the to-dos in the field are discussed.

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.

Guanine-rich nucleic acids can fold into G-quadruplexes, secondary structures implicated in important regulatory functions at the genomic level in humans, prokaryotes and viruses. The remarkably high guanine content of the Herpes Simplex Virus-1 (HSV-1) genome prompted us to investigate both the presence of G-quadruplex forming sequences in the viral genome and the possibility to target them with G-quadruplex ligands to obtain anti-HSV-1 effects with a novel mechanism of action. Using biophysical, molecular biology and antiviral assays, we showed that the HSV-1 genome displays multiple clusters of repeated sequences that form very stable G-quadruplexes. These sequences are mainly located in the inverted repeats of the HSV-1 genome. Treatment of HSV-1 infected cells with the G-quadruplex ligand BRACO-19 induced inhibition of virus production. BRACO-19 was able to inhibit Taq polymerase processing at G-quadruplex forming sequences in the HSV-1 genome, and decreased intracellular viral DNA in infected cells. The last step targeted by BRACO-19 was viral DNA replication, while no effect on virus entry in the cells was observed. This work, presents the first evidence of extended G-quadruplex sites in key regions of the HSV-1 genome, indicates the possibility to block viral DNA replication by G-quadruplex-ligand and therefore provides a proof of concept for the use of G-quadruplex ligands as new anti-herpetic therapeutic options.

Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy.

Our findings strongly support the activity of BRACO-19 at the viral G-quadruplex level and therefore strengthen the use of viral G-quadruplexes as new anti-HIV-1 targets.