Periodic Reporting for period 4 - INTEGHER (Integration of herpesvirus into telomeres: From the mechanism of genome integration and mobilization to therapeutic intervention)
Période du rapport: 2020-10-01 au 2022-03-31
In the ERC project INTEGHER, novel techniques were established and used to explore this integration mechanism and to develop therapeutic approaches. Specifically, we 1) determined the fate of the HHV-6 genome during latency by developing a novel system that allows imaging of the virus genome in living cells and elucidated epigenetic changes of the HHV-6 genome during integration and reactivation, 2) identified viral and cellular factors that drive virus genome integration and reactivation, using recombinant viruses, drugs and CRISPR/Cas9 gene editing and 3) used gene editing tools to eliminate the virus genome integrated in host chromosomes. Our proposal utilized state-of-the-art technologies and pioneered new approaches, particularly with regard to visualization and excision of virus genomes in latently infected cells that are also present in (bone marrow) transplants. Altogether, our studies shed light on the mechanism of herpesvirus integration and reactivation and provided a new tool for therapeutic excision of virus genomes in the clinics.
In the first aim, we developed a tool that allows visualization of herpesvirus genomes in living cells. Our experiments revealed that the replication centers, which form in the nucleus, are very dynamic and appear rapidly after infection. In addition, we could detect the integrated virus genome in the quiescent phase of infection (latency), where one or more individual viral genomes were detected. We also investigated the silencing of the HHV-6 genome during integration and could show that the virus genome is completely silenced within a matter of days. Furthermore, we demonstrated that the virus genome is silenced on an epigenetic level and highly compacted, preventing the expression of viral genes.
In the second aim, we assessed which viral and cellular factors facilitate HHV-6 integration. We could show that telomere sequences in the HHV-6 genome facilitate the integration into human telomeres. This study provided the first molecular evidence on the HHV-6 integration mechanism and was published in PLoS Pathogens. In addition, we addressed the role of viral and cellular proteins in the integration process. We could prove that the putative viral integrase U94 is dispensable for HHV-6 integration. Furthermore, we could demonstrate that the U70 protein of HHV-6 enhances recombination via the single strand annealing (SSA) pathway. However, this protein is also not essential for integration. On the cellular side, we could reveal that telomerase activity, and the host proteins PML and TRF-2 are required for efficient integration of the virus into host telomeres. Moreover, our work revealed that the ND10 complex suppresses HHV-6 replication and reactivation.
In the third aim, we established a tool to eliminate the integrated HHV-6 genome from cells using the CRISPR/Cas9 system. Prior to excision, we characterized the HHV-6 integration sites to obtain insights into the target region. We developed a novel whole-genome optical nanopore mapping approach that provided a high-resolution map of the integration site for multiple iciHHV-6 patients. Using this knowledge, we optimized our CRISPR/Cas9 approach to increase the efficiency of HHV-6 genome excision. By increasing the number of target sites for the excision, we could drastically increase the excision efficiency and eliminate the virus in most cells (Aimola, et al. in preparation). We also successfully used this approach on iciHHV-6 patient cells, providing a therapeutic approach for the excision of virus genomes in the clinics.