G-quadruplexes in the herpes simplex virus type 1 and measles virus genomes: new antiviral targets

G-quadruplexes (G4s) are four-stranded secondary structures formed by guanine-rich nucleic acids; they are implicated in important functions as epigenetic regulators at the genomic level in humans, prokaryotes and viruses. They act as silencers in the promoter regions of human genes and, moreover, they have been proposed to be directly involved in gene regulation at the transcription level. Since the Herpes Simplex Virus Type 1 (HSV-1) genome is significantly rich in guanines (68%) and its G4 sequences are massively present during viral replication, we aimed at investigating the possibility to target the HSV-1 G4s by a core extended naphthalene diimide (c-exNDI) G4–ligand to obtain anti HSV-1 effects with an innovative mechanism of action. Biophysical and biomolecular analysis proved that c-exNDI stabilized the HSV-1 G4s in a concentration-dependent manner and was able to inhibit Taq polymerase processing at G4 forming sequences. Additionally, we proved that c-exNDI preferentially recognized HSV-1 G4s over cellular telomeric G4s, the most represented G4s within cells. Treatment of HSV-1 infected cells with c-exNDI at low nanomolar concentrations induced significant virus inhibition with low cytotoxicity. The mechanism of action was ascribed to a G4-mediated inhibition of viral DNA replication, with consequent impairment of viral gene transcription. Our data suggested that the observed potent antiviral activity and low cytotoxicity mainly depend on a combination of c-exNDI affinity for HSV-1 G4s and their massive presence during infection. Since the vast majority of G4-ligands share the common characteristic of a large aromatic surface which makes them not well druggable, we then aimed at investigating the ability of an in-house library of compounds, previously designed to target promoters in cancer cells, to bind and stabilize HSV-1 G4s and to exert an antiviral activity against HSV-1. We demonstrated that all compounds of the library were able to bind and stabilize HSV-1 G4s in a concentration-dependent manner with different affinities and, again, to induce polymerase stalling because of a steric hindrance linked to the G4 stabilization. Treatment of HSV-1 infected cells with all compounds of the library showed a conspicuous decrease in viral production in the low nanomolar range. Two of the four compounds were also only mildly cytotoxic and thus showed promising selectivity indexes (SI). The mechanism of action, once again, was ascribed to the G4-mediated inhibition of viral DNA replication. Since all compounds of the library possess (i) promising chemical features (i.e their small size, smaller than other well known G4-ligands such as B-19 and NDIs) and (ii) potent activities in the low nanomolar range with no cytotoxicity, this makes them suitable for future development as novel anti HSV-1 G4-ligands with more drug-like characteristics than previous developed G4-ligands, and as promising compounds for the treatment of ACV-resistant herpetic infections. In the third part of this study, by using a pull-down approach, we aimed at identifying viral and cellular proteins able to interact specifically with G4 structures within HSV-1 infected cells. We preliminarily identified both cellular and viral proteins directly implicated in replication and transcription processes. Concerning cellular proteins, we identified hnRNPU, nucleolin, nucleophosmin and histone proteins (11 and 12), some of them being already reported for their ability to bind G4s during replication. Identification of histone proteins opens the possibility that G4s are involved in the latent maintenance of the viral genome. Besides viral proteins we identified the transcription factor ICP4, the single-stranded DNA binding protein ICP8, PAP1 polymerase processing factor and VP5. The identification of the two transcription factors and of the polymerase processing factor confirmed the hypothesis of a direct involvement of G4s in the viral genome replication and transcription and the identification of the major capsid protein VP5 suggested a possible role of G4s as a driving force for viral genome encapsidation in the virions assembly process. Finally, by investigating the negative-stranded RNA genome of Measles Virus (MV) we demonstrated the presence of seven G4-putative folding sequences. Six of these sequences displayed the ability to fold into G4 structures while one showed a hairpin-like conformation. All sequences were bound and stabilized by B-19 G4-ligand and treatment of MV infected cells with B-19 induced a pronounced reduction in the viral genome accumulation. Since (i) G4s are well conserved and distributed within different viral genomes (as described by our group in HIV-1, HSV-1, and MV) because of their central roles in regulating biological processes and (ii) different G4-ligands have been demonstrated to be active against different viruses (i.e B-19, c-exNDI and G4-ligands of an in-house library in this thesis) with a G4-mediated inhibition of viral DNA replication, we can strongly encourage and confirm the hypothesis using G4s as novel broad range drug targets against different etiological agents and G4-ligands as novel innovative therapeutic molecules.