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

Abstract

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.

Visualization of DNA G-quadruplexes in herpes simplex virus 1-infected cells

“…We have previously shown that at least four G-rich repeated sequences in the HSV-1 genome can fold into G4 structures in vitro (17). Three sequences feature four GGGG-tracts, while one sequence has four GGG-tracts, displaying the ability to form multiple monomolecular 4- or 3-tetrad stacked G4, respectively (Table 1).…”

“…The herpes simplex virus-1 (HSV-1) genome has a very high GC content (68%) which peaks at 84.7% GC in simple sequence repeats (SSRs) (16). Recently we provided evidence for the presence of very stable G4-forming regions located in the HSV-1 inverted repeats (17). In particular, multiple conserved and extended clusters of G4 forming sequences were observed, covering about 2,000 bp of the 152,000 bp-viral genome.…”

“…Both in the case of HSV-1 and HIV-1, treatment of infected cells with G4 ligands greatly impaired viral infectivity (13,15,17); in particular, treatment with BRACO-19 stabilized G4s in the HSV-1 genome and inhibited viral replication (17). …”

Genome-wide analysis of G-quadruplexes in herpesvirus genomes

“…Recently, Artusi et al, have reported the presence of G-quadruplex repeats in the HHV-1 genome [17]. It is well known that G-quadruplex structures are very stable.…”

“…Studies on G-quadruplexes in virus genomes are limited. A few studies show functional roles of selected RNA or DNA G-quadruplex motifs present in the virus genome in vitro [13–17] or describe the genomic location of G-quadruplexes in virus genomes [18]. …”