Periodic Reporting for period 1 - G4-KuRE (Roles of G-quadruplexes in Kaposi’s sarcoma-associated herpesvirus latent REplication)
Berichtszeitraum: 2022-06-01 bis 2024-05-31
Herpesviruses have the ability of maintaining themselves in a life-long state of latency after infecting an individual. Some of these viruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV) also behave as oncogenic viruses, as their latent infection constitutes a risk to develop cancer, particularly for immunocompromised individuals. This represents a considerable social and economic burden for low-income countries, but also 2-5% of cancer incidences in Europe can be attributed to oncogenic viruses. Current strategies aim at defining cures for the cancers derived from oncogenic virus infections. A possibly more effective approach would be, however, to identify efficient ways to block the virus from replicating in the infected cells. To tackle this problem the G4-KuRE project achieved two main goals. First, with a novel approach using protein extracts coming from frog eggs, it identified specific DNA structures in the genome of KSHV that are susceptible to being targeted to block viral replication. Second, it identified drugs that can further stabilise those structures and eukaryotic proteins that contribute to their efficient replication. These results constitute a very solid base for the further development of an effective antiviral strategy against KSHV.
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.
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.
Based on the identification of compounds able to significantly block replication of the TRs of KSHV and of proteins that, if inhibited, could exacerbate this blockage effect, further immediate studies will allow these results to be fully exploited towards the development of an effective antiviral strategy against latent KSHV.
First, experiments in KSHV infected cells will indicate if treatment with the identified compounds effectively blocks replication of the virus. Furthermore, concomitant down regulation of the key proteins identified will likely exacerbate this antiviral effect.
Finally, further optimisations will be needed to identify the most virus specific combined treatments, which exhibits the lowest toxicity for the cells. These results will be an excellent base for a company-based implementation of an antiviral approach.
First, experiments in KSHV infected cells will indicate if treatment with the identified compounds effectively blocks replication of the virus. Furthermore, concomitant down regulation of the key proteins identified will likely exacerbate this antiviral effect.
Finally, further optimisations will be needed to identify the most virus specific combined treatments, which exhibits the lowest toxicity for the cells. These results will be an excellent base for a company-based implementation of an antiviral approach.