Paragraph: In herpes simplex virus 1 (HSV-1)-infected cells, G4s are massively present during viral replication. We here aimed at investigating the possibility to target the HSV-1 G4s by a core extended naphtalene diimide (c-exNDI) G4 ligand. Biophysical and biomolecular analysis proved that c-exNDI stabilized the HSV-1 G4s in a concentration dependent manner. In MS competition assays, c-exNDI preferentially recognized HSV-1 G4s over cellular telomeric G4s, the most represented G4s within cells; other less abundant cellular G4s were also recognized. Treatment of HSV-1 infected cells with c-exNDI at low nanomolar concentrations induced significant virus inhibition with no cytotoxicity. The mechanism of action was ascribed to G4-mediated inhibition of viral DNA replication, with consequent impairment of viral genes transcription. Our data suggest 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. HSV-1 G4s may thus represent new effective antiviral targets: the fact that no current antiherpetic drug exploits them and their presence at the viral genome, responsible for both active and latent HSV infections, makes them particularly attracting. G-quadruplexes (G4s) are nucleic acids secondary structures that may form in single-stranded G-rich sequences under physiological conditions 1 . Four Gs bind via Hoogsteen-type hydrogen bonds base-pairing to yield G-quartets, which stack to form the G4. The presence of K + cations specifically supports G4 formation and stability 2 . Based on the strand orientation, G4s can adopt three main topologies: parallel, antiparallel, and hybrid-type structures. In eukaryotes, G4s have been shown to be involved in key regulatory roles, including transcriptional regulation of gene promoters and enhancers, translation, chromatin epigenetic regulation, DNA recombination 3-7 . Expansion of G-quadruplex-forming motifs has been associated with relevant human neurological disorders 4, 8, 9 . Formation of G4s in vivo has been consolidated by the discovery of cellular proteins that specifically recognize G4s 10, 11 and the development of G4 specific antibodies 12, 13 . Recently, the presence of G4s in viruses and their involvement in virus key steps has been provided 14 . G4s have been implicated in pathogenic mechanisms of the human immunodeficiency virus, where functionally significant G4s have been identified 5, 11, 15-17 and stabilized by G4 ligands with consequent antiviral effects 5, 18, 19 . G4s have been reported in the SARS coronavirus 20 , the human papilloma, Zika, Ebola and hepatitis C virus genome 21-24 . Among herpesviruses, RNA G4s have been implicated in the regulation of DNA replication and translation of the Epstein-Barr virus 25, 26 . We have shown that the herpes simplex virus 1 (HSV-1) possesses several repeats of important G4-forming sequences, which could be stabilized by a G4 ligand with inhibition of viral DNA replication 27 . In addition, HSV-1 G4s, visualized with the aid of a G4-specific antibody in infected cells 12 , were shown to form massively in the cell nucleus, peak during viral replication and localize according to the viral genome intracellular movements 28 . The involvement of G4 structures in several human diseases propelled the development of small molecules directed against G4s 7, 29 . However, only very few have been tested against viruses, i.e. Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 Scientific RepoRts | 7: 2341 | 
 Title: A core extended naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication OPEN
Output: True