Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - INTEGHER (Integration of herpesvirus into telomeres: From the mechanism of genome integration and mobilization to therapeutic intervention)

Teaser

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...

Summary

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 patient 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 will be established and used to explore this integration mechanism and to develop therapeutic approaches. Specifically, we will 1) determine 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 elucidate epigenetic changes of the HHV-6 genome during integration and reactivation, 2) identify viral and cellular factors that drive virus genome integration and reactivation, using recombinant viruses, drugs and CRISPR/Cas9 gene editing and 3) Use gene editing tools to eliminate the virus genome integrated in host chromosomes in vitro and in an in vivo model. Our proposal utilizes state-of-the-art technologies and pioneers 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, these studies will define the mechanism of herpesvirus integration and reactivation and will provide new tools for therapeutic excision of virus genomes in the clinics.

Work performed

In the first period of the ERC starting grant INTEGHER, we successfully addressed the aims according to the time line proposed in the project and laid the foundation for the final period as outlined below.
In the first aim, we developed a tool that allows visualization of herpesvirus genomes in living cells. Using our new imaging system, we were able to track replicating virus genomes in the nucleus of infected cells. These experiments revealed that the replication centers that form in the nucleus are very dynamic and form rapidly after infection. In addition, we could detect the virus genome in the quiescent phase of infection (latency), where one or more individual virus genomes were detected. We are now using this system to visualize the integration process and virus reactivation as proposed in the ERC grant. In addition, we investigated the chromatinization of the HHV-6 integrated genome in host telomeres and the expression of its genes. We could demonstrate that the virus genome is heavily silenced and highly compacted preventing the expression of viral genes. This work has been submitted to the “Journal of Virology” and is currently under review.
In the second aim, we assessed which viral and cellular factors facilitate HHV-6 integration. We could demonstrate that telomere sequences in the HHV-6 genome facilitate the integration. This study provided the first molecular evidence on the HHV-6 integration mechanism and was recently published in PLoS Pathogens. In addition, we addressed the role of viral and cellular proteins in the integration process. We could demonstrate 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 demonstrate that inhibition of several cellular pathways reduces the integration efficiency in the absence of the putative viral recombinases. Some of these studies were recently published in the journals JGV and Viruses, while others will be submitted soon. For the identification of factors that suppress reactivation that will be addressed in the second period, we generated cell lines harboring the integrated HHV-6 genome and express a green fluorescent protein (GFP) in > 95% of the cells up stimulation with drugs commonly used for herpesvirus reactivation. This will provide the basis for the experiments proposed for the second period.
In the third aim, we established a tool to eliminate the integrated HHV-6 genome from cells. We optimized the gene editing tools to increase the efficiency of integration. By increasing the number of target sites for the excision, we could drastically increase the excision efficiency and eliminate the virus in most cells. We will use this approach on clinical samples and use it for our in vivo model for herpesvirus integration in the second period of the ERC grant.

Final results

Aside from successfully addressing the proposed aims, we also made progress beyond the state of the art and expected results of the project. In the first aim, we are developing an approach for the visualization of the virus genome, that is independent of the generation of recombinant viruses. This approach could also be applied to other viruses in the future and provide a great tool for the field. For the third aim, we established a gene editing approach that allows multiplexing of 10 target sites at the ends of the HHV-6 genome for its excision. We will use this approach also for other aims in the second period of the ERC project.