In the initial phases of the project a detailed analysis of the genome of KSHV has been performed, with a particular focus on a repeated region of the viral genome named terminal repeats (TRs), which were known to be highly rich in cytosines and guanines. Such regions are prone to assume a non-canonical conformation known as G-quadruplexe (G4). Importantly, G4s are more difficult to replicate than canonically paired bases. Indeed, by bioinformatic and biophysical analyses I identified several G4s in the TR region.
I further analysed the replication of these particular regions implementing a new approach to study replication of complex viral element in vitro. This approach relies on the use of frog egg extracts, which have the remarkable ability to recapitulate eukaryotic DNA replication in a test tube. After validating the use of this system to recapitulate latent viral replication, I observed that replication of the TRs was slower that other DNA sequences. Importantly, I could show that molecules such as Pyridostatin of PhenDC3, which stabilise G4 structures, further blocked TR replication in vitro.
Moreover, by mass spectrometry-based approaches I Identified eukaryotic proteins and pathways involved in the efficient replication of the TRs. Tests that are currently being performed will indicate if blocking the activity of any of these proteins will specifically block replication of the TRs.
Overall, the action identified the G4s in the TRs as a novel vulnerability in KSHV. Moreover, it characterised both synthetic molecules and eukaryotic proteins that can be used to further develop an antiviral approach against KSHV latency.
