The Herpes Simplex Virus-1 genome contains multiple clusters of repeated G-quadruplex: Implications for the antiviral activity of a G-quadruplex ligand

• Large clusters of repeated G-quadruplex folding sequences were identified in the HSV-1 genome.
• BRACO-19, a G-quadruplex ligand, was able to bind and stabilize the HSV-1 G-quadruplexes.
• Stabilization of HSV-1 G-quadruplexes by BRACO-19 inhibited DNA polymerase processing and viral DNA replication.
• BRACO-19 diminished virus production in infected cells.
• HSV-1 G-quadruplex may be regarded as alternative and innovative targets for antiviral therapy.

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.

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