Characterization and targeting of G-quadruplexes in the genome of Kaposi's sarcoma-associated herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus capable of establishing life-long latent infections in host cells, ultimately resulting in Kaposi’s sarcoma, an incurable cancer affecting immunocompromised patients. Maintenance of KSHV latent infection requires binding of the viral protein Latency-Associated Nuclear Antigen (LANA) both to the host cell chromosomes and to the viral non-coding terminal repeat (TR) sequences, highly guanine-cytosine-rich repeats that comprises about 20% of KSHV genome. Given their sequence, it has been speculated that TRs can fold into G-quadruplexes (G4s), secondary DNA structures whose role in KSHV latent replication remains poorly understood. The purpose of this work was to identify and characterize G4s within the TR region of KSHV, with the long-term goal to understand their role in the latent replication of KSHV and to target them in order to block replication of the latent virus. To this aim, we initially performed a bioinformatic analysis and identified putative G4-forming sequences (PQSs) within the TR. We then confirmed their folding in vitro by circular dichroism spectroscopy (CD) and PCR stop assay, showing a significant effect of G4 ligands in stabilizing these structures. We further characterized PQSs by CD thermal unfolding experiments and we finally identified several G4-forming sequences at different pausing sites. Altogether, these results prove that putative G4-forming sequences within the TR can fold into G4s and highlight how G4 structures can be further investigated as a potential target for the treatment of KSHV latent infection.