G-quadruplexes and G-quadruplex ligands: targets and tools in antiviral therapy

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

Stable and Conserved G-Quadruplexes in the Long Terminal Repeat Promoter of Retroviruses

Retroviruses infect almost all vertebrates, from humans to domestic and farm animals, from primates to wild animals, where they cause severe diseases, including immunodeficiencies, neurological disorders, and cancer. Nonhuman retroviruses have also been recently associated with human diseases. To date, no effective treatments are available; therefore, finding retrovirus-specific therapeutic targets is becoming an impelling issue. G-Quadruplexes are four-stranded nucleic acid structures that form in guanine-rich regions. Highly conserved G-quadruplexes located in the long-terminal-repeat (LTR) promoter of HIV-1 were shown to modulate the virus transcription machinery; moreover, the astonishingly high degree of conservation of G-quadruplex sequences in all primate lentiviruses corroborates the idea that these noncanonical nucleic acid structures are crucial elements in the lentiviral biology and thus have been selected for during evolution. In this work, we aimed at investigating the presence and conservation of G-quadruplexes in the Retroviridae family. Genomewide bioinformatics analysis showed that, despite their documented high genetic variability, most retroviruses contain highly conserved putative G-quadruplex-forming sequences in their promoter regions. Biophysical and biomolecular assays proved that these sequences actually fold into G-quadruplexes in physiological concentrations of relevant cations and that they are further stabilized by ligands. These results validate the relevance of G-quadruplexes in retroviruses and endorse the employment of G-quadruplex ligands as innovative antiretroviral drugs. This study indicates new possible pathways in the management of retroviral infections in humans and animal species. Moreover, it may shed light on the mechanism and functions of retrovirus genomes and derived transposable elements in the human genome

Genome-wide mapping of i-motifs reveals their association with transcription regulation in live human cells

Lay Summary Among the secondary structures alternative to the DNA double helix, i-Motifs (iMs) and G-quadruplexes (G4s) are four-stranded non-canonical nucleic acid structures that form in cytosine- and guanine-rich regions, respectively. Because iMs fold in vitro under acidic conditions, they were long thought to form only in vitro. We now show that iMs, like G4s, form in live human cells mainly at gene promoters in open chromatin. iMs that are unstable in vitro still form in cells. iMs and G4s are cell-type specific and associated with increased transcription; however, transcript levels are remarkably different: low for iMs and high for G4s, indicating their distinct activity as regulators of the cell transcriptome. The iM/G4 interplay may represent a novel therapeutic target in disease.i-Motifs (iMs) are four-stranded DNA structures that form at cytosine (C)-rich sequences in acidic conditions in vitro. Their formation in cells is still under debate. We performed CUT & Tag sequencing using the anti-iM antibody iMab and showed that iMs form within the human genome in live cells. We mapped iMs in two human cell lines and recovered C-rich sequences that were confirmed to fold into iMs in vitro. We found that iMs in cells are mainly present at actively transcribing gene promoters, in open chromatin regions, they overlap with R-loops, and their abundance and distribution are specific to each cell type. iMs with both long and short C-tracts were recovered, further extending the relevance of iMs. By simultaneously mapping G-quadruplexes (G4s), which form at guanine-rich regions, and comparing the results with iMs, we proved that the two structures can form in independent regions; however, when both iMs and G4s are present in the same genomic tract, their formation is enhanced. iMs and G4s were mainly found at genes with low and high transcription rates, respectively. Our findings support the in vivo formation of iM structures and provide new insights into their interplay with G4s as new regulatory elements in the human genome

A dynamic i-motif with a duplex stem-loop in the long terminal repeat promoter of the HIV-1 proviral genome modulates viral transcription

I-motifs are non-canonical nucleic acids structures characterized by intercalated H-bonds between hemi-protonated cytosines. Evidence on the involvement of i-motif structures in the regulation of cellular processes in human cells has been consistently growing in the recent years. However, i-motifs within non-human genomes have never been investigated. Here, we report the characterization of i-motifs within the long terminal repeat (LTR) promoter of the HIV-1 proviral genome. Biophysical and biochemical analysis revealed formation of a predominant i-motif with an unprecedented loop composition. One-dimensional nuclear magnetic resonance investigation demonstrated formation of three G-C H-bonds in the long loop, which likely improve the structure overall stability. Pull-down experiments combined with mass spectrometry and protein crosslinking analysis showed that the LTR i-motif is recognized by the cellular protein hnRNP K, which induced folding at physiological conditions. In addition, hnRNP K silencing resulted in an increased LTR promoter activity, confirming the ability of the protein to stabilize the i-motif-forming sequence, which in turn regulates the LTR-mediated HIV-1 transcription. These findings provide new insights into the complexity of the HIV-1 virus and lay the basis for innovative antiviral drug design, based on the possibility to selectively recognize and target the HIV-1 LTR i-motif

Antiviral Activity of the G-Quadruplex Ligand TMPyP4 against Herpes Simplex Virus-1

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro; in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways

82. Cftr Gene Targeting in Murine ES Cells Mediated by the SFHR Technique

Small Fragment Homologous Recombination (SFHR)-mediated targeting is a gene therapy strategy where a specific genomic locus is modified through a target exchange between a small DNA fragment (SDF) and genomic DNA. Here we demonstrate that SFHR can stably introduce a 3-bp deletion (corresponding to |[Delta]|F508) within Cftr (Cystic Fibrosis Transmembrane Conductance Regulator) locus in the genome of mouse embryonic stem (ES) cells. SDFs (about 6.4|[times]|105 molecules per cell) carrying the |[Delta]|F508 mutation were transfected by nucleofection protocol. About 12% of transcript corresponding to deleted allele was detected and about 60% of the electroporated cells no longer had measurable CFTR-dependent chloride efflux. The CFTR activity was also analyzed by measuring the chloride efflux by the fluorescence microscopy-coupled digital video imaging system in each ES cell colony, previously loaded with MQAE, a chloride sensitive dye. An average of 4-6 regions for each cell colony was analysed to verify the genotypic homogeneity of each colony. In fact all regions examined in each colony showed a similar significant chloride efflux after PKA activation. Moreover on twelve electroporated ES colonies analysed, eight were successfully mutated (Cl- efflux not significantly different from zero) while four colonies showed Cl-efflux CFTR-dependent not significantly different from the untreated ones

Epithelial ovarian cancer is infiltrated by activated effector T cells co-expressing CD39, PD-1, TIM-3, CD137 and interacting with cancer cells and myeloid cells

IntroductionDespite predicted efficacy, immunotherapy in epithelial ovarian cancer (EOC) has limited clinical benefit and the prognosis of patients remains poor. There is thus a strong need for better identifying local immune dynamics and immune-suppressive pathways limiting T-cell mediated anti-tumor immunity.MethodsIn this observational study we analyzed by immunohistochemistry, gene expression profiling and flow cytometry the antigenic landscape and immune composition of 48 EOC specimens, with a focus on tumor-infiltrating lymphocytes (TILs).ResultsActivated T cells showing features of partial exhaustion with a CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ surface profile were exclusively present in EOC specimens but not in corresponding peripheral blood or ascitic fluid, indicating that the tumor microenvironment might sustain this peculiar phenotype. Interestingly, while neoplastic cells expressed several tumor-associated antigens possibly able to stimulate tumor-specific TILs, macrophages provided both co-stimulatory and inhibitory signals and were more abundant in TILs-enriched specimens harboring the CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ signature.ConclusionThese data demonstrate that EOC is enriched in CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ T lymphocytes, a phenotype possibly modulated by antigen recognition on neoplastic cells and by a combination of inhibitory and co-stimulatory signals largely provided by infiltrating myeloid cells. Furthermore, we have identified immunosuppressive pathways potentially hampering local immunity which might be targeted by immunotherapeutic approaches