Periodic Reporting for period 4 - INTEGHER (Integration of herpesvirus into telomeres: From the mechanism of genome integration and mobilization to therapeutic intervention) Reporting period: 2020-10-01 to 2022-03-31 Summary of the context and overall objectives of the project Herpesviruses cause serious diseases in humans and animals and become dormant (known as latency) in the host for life. We and others have recently identified a novel mechanism of latency that allows some herpesviruses to integrate their complete genetic material into the ends of host chromosomes. One of these herpesviruses is human herpesvirus 6 (HHV-6), which causes a three-day fever (Roseola Infantum) in infants that can be accompanied by inflammation of the brain (encephalitis). Intriguingly, HHV-6 can also integrate its genome in sperm or egg cells (germ cells). This has resulted in individuals that harbor the integrated virus in every cell of their body and pass it on to their offspring. About one percent of the world's population have this condition termed inherited chromosomally integrated HHV-6 (iciHHV-6); however, the biological consequences remain poorly understood. HHV-6 can reactivate in iciHHV-6 patients and from latency, which is associated with a number of diseases including seizures, encephalitis, and graft rejection in transplant patients. Until now, we lack a comprehensive framework to investigate the molecular mechanisms of HHV-6 integration and to assess the clinical consequences of its reactivation.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. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In the ERC starting grant INTEGHER, we successfully addressed the aims as proposed in the project and already published 16 manuscripts in high impact journals including Nature, Nature Biotechnology, MBE and PNAS. Additional manuscripts are currently in preparation. Beyond that, the data obtained in the INTEGHER laid the foundation for current and future projects and grants. 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. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In addition to successfully addressing the proposed aims, we made progress beyond the state of the art. For example, we developed a novel whole-genome optical nanopore mapping approach based on the Bionano Saphyr technology to map the integration sites of the virus genome in iciHHV-6 patients, which was recently published in PNAS (Wight, et al. 2020). We could also demonstrate that integration of HHV-6 into the germline already occurred before humans moved out of Africa (Aswad, et al. 2021). This ancient integration resulted in distinct clades of the virus with almost identical viral sequences and the same integration site. Furthermore, more recent integration events were identified, suggesting that this process is a still ongoing (Aswad, et al. 2021). In collaboration with Prof. Ewers (Freie Universität Berlin), we developed a novel approach to visualize the human herpesvirus 6 (HHV-6) genome by super-resolution microscopy in the nucleus of infected cells, which was recently published in Nature Biotechnology (Geertsema, et al. 2020). In another collaboration with Prof. Dölken and Dr. Prusty at the Julius-Maximilians-Universität Würzburg, we discovered that the HHV-6 miR-aU14 can trigger virus reactivation from latency. miR-aU14 achieves this by inhibiting the processing of specific miR-30 family members, which in turn triggers a profound disruption of the mitochondrial architecture. This process impaired the induction of type I interferons and was shown to be necessary for both productive infection and HHV-6 reactivation. This exciting discovery was recently published in the Journal Nature (Hennig, et al. 2022). HHV-6 integration - Annual Review of Virology 2014 1:1, 215-235
