Genome-wide mapping of G-quadruplexes in human cells infected with Herpes Simplex Virus-1 and their impact on the cell transcriptome

The Herpes Simplex Virus-1 (HSV-1) is a double-stranded DNA virus that causes life-long infections in humans and establishes latency in the neuronal cells of the host. HSV-1 is associated with a variety of symptoms, from asymptomatic infections to life-threatening complications, like meningitis or encephalitis, especially in immunocompromised patients. With an estimated 70% of the world’s population infected, current drugs only treating symptoms and the emergence of resistant strains, new therapeutic approaches are urgently needed. However, several features of HSV-1 biology and viral cycle remain unclear. There is increasing evidence that viruses modify chromatin organisation and three-dimensional DNA structures to exploit the cell for their own replication. Additionally, essential cellular processes rely on the DNA’s topological state. It is widely recognised that DNA adopts various sequence-dependent secondary structures that differ from the traditional Watson-Crick double helix. Among these, G-quadruplexes (G4s), which are four-stranded non-canonical secondary structures arising from guanine-rich sequences, are the most abundant and thoroughly studied non-canonical structures that have been shown to be involved in various cellular processes. The development of specific G4-antibodies has allowed G4-mapping at the genome-wide level in several cell lines, demonstrating G4 enrichment in human gene promoters and their correlation with increased gene transcription. With these premises, the aim of this thesis was to investigate the impact of HSV-1 infection on the host G4s. We employed CUT&Tag, an innovative chromatin immunoprecipitation technique, coupled with sequencing using the anti-G4 antibody (BG4) in human cells infected with HSV-1. We monitored changes in the host G4-landscape during the early stages of the viral life cycle, up to 5 hours post-infection. We observed that HSV-1 mainly induces the accumulation of new G4s at gene promoters, but it also induces the disappearance of existing G4s in these regions. Since promoters are the main regulators of gene expression, we investigated how G4s affect transcription by integrating CUT&Tag data with the expression levels of each transcript. We focused on the genes that showed the greatest difference in expression levels in response to changes in the G4 profile at the promoter. We found that these genes were mainly involved in cellular responses to stress, RNA metabolism, and translation pathways. Among the most significant hits, we identified genes responsible for pathogen recognition (TRIM family proteins), regulation of RNA Pol II (ETV3 and JUNB), and stress response (HSPA6). This work provides new insights into the association between G4s and transcriptional regulation in response to viral infections. It opens the possibility for the identified genes to become potential therapeutic targets for HSV-1 treatments.