Endogenous Human Herpesvirus: Germ line integration and effects on host cell and organism

Project Information

ENDo-HERPES

Grant agreement ID: 101087480

DOI

10.3030/101087480

EC signature date 13 March 2023

Start date 1 September 2023

End date 31 August 2028

Funded under

• European Research Council (ERC)

Total cost

€ 2 000 000,00

EU contribution

€ 2 000 000,00

Coordinated by

FREIE UNIVERSITAET BERLIN
Germany

Periodic Reporting for period 1 - ENDo-HERPES (Endogenous Human Herpesvirus: Germ line integration and effects on host cell and organism)

Reporting period: 2023-09-01 to 2026-02-28

Summary of the context and overall objectives of the project

Viruses have played an important role in human evolution, and some have even become a permanent part of our DNA. The ENDo-HERPES project explores one such example — a herpesvirus called human herpesvirus 6 (HHV-6) — which has integrated its genetic material into human chromosomes and is now inherited by around 80 million people worldwide. This remarkable condition, known as inherited chromosomally integrated HHV-6 (iciHHV-6), means that the virus is present in every cell of the affected individual and can be passed on to future generations. Although HHV-6 is common and usually harmless, its reactivation from the integrated state has been linked to several severe diseases, including seizures, encephalitis, heart failure, and organ transplant rejection. Yet, we still know very little about how and why these reactivations occur, or how the integrated virus affects human health.
ENDo-HERPES seeks to answer these questions using cutting-edge genomic, molecular, and cellular technologies. The project will first determine which of the distinct groups of integrated viral genomes are still functional and capable of producing infectious virus. It will then identify where in human chromosomes the virus has integrated and how this process occurred. Finally, it will investigate how the presence of the viral genome influences host cells and whether age-related shortening of chromosome ends, called telomeres, can trigger the virus to reactivate. These studies combine advanced techniques such as CRISPR/Cas9 genome editing, long-read nanopore sequencing, and high-resolution imaging to uncover the intricate relationship between the virus and the human genome.
By revealing the biological mechanisms behind HHV-6 integration and reactivation, ENDo-HERPES will help clarify whether the virus merely coexists with us or actively contributes to disease. This knowledge will likely lead to new diagnostic tools to identify individuals at risk and guide the development of treatments that prevent or control virus reactivation. Beyond virology, the findings will provide insights into telomere biology, human evolution, and the long-term consequences of viral DNA embedded in our genome.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During the first reporting period, significant progress was made across all project aims. In Aim 1a, we successfully developed and optimized a transformation-associated recombination (TAR)-based strategy for transferring entire human herpesvirus 6 (HHV-6) genomes into a reverse genetic system. To ensure fidelity, we established an amplification-free approach using Cas9-mediated cleavage of high-molecular-weight viral DNA combined with optimized TAR vectors. This enabled the generation of the first complete genetic systems for endogenous HHV-6A and HHV-6B (eHHV-6), as well as standard laboratory strains. In Aim 1b, BAC-based virus genomes were reconstituted in appropriate T-cell lines. Early results revealed strain- and cell line-specific differences in viral behavior, which are being further characterized. In Aim 2a, a Cas9-targeted nanopore sequencing method was refined to map eHHV-6 integration sites at high resolution. This approach produced long sequencing reads spanning entire virus–host junctions and allowed precise identification of chromosomal integration sites, which were validated by fluorescence in situ hybridization (FISH). In Aim 2b, this technology was applied to elucidate the mechanism of HHV-6 integration. Using mutant viruses lacking specific telomeric repeat motifs, we demonstrated that the telomeric repeats are crucial for stable integration into host telomeres. In Aim 3a, we established and validated a CRISPR/Cas9-based system for precise excision of integrated eHHV-6 genomes, providing a platform to assess the impact of the virus on host gene expression in the next reporting period. Finally, in Aim 3b, we investigated whether telomere shortening triggers viral reactivation. Using telomerase knockout cells, we observed virus reactivation during telomere crisis, indicating a direct link between telomere erosion and virus reactivation. Together, these achievements represent major methodological and conceptual advances, providing new tools and evidence to understand how endogenous HHV-6 persists, integrates, and reactivates, thereby laying the groundwork for identifying its contribution to human disease.

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)

The ENDo-HERPES project delivered major advances beyond the state of the art by developing innovative tools to study large viral genomes and their interaction with human DNA. An important achievement was the establishment of a Cas9-assisted, amplification-free TAR cloning system that enables accurate capture of entire HHV-6 genomes without introducing mutations—a method adaptable to other complex DNA viruses. The team also optimized virus reconstitution, reducing the recovery time from months to days. Another major innovation was the Cas9-targeted nanopore sequencing approach, which enables complete mapping of virus–host junctions and telomeric integration sites at single-molecule resolution. In addition, the first CRISPR/Cas9 system to excise integrated viral genomes was established allowing the removal of the endogenous virus. Finally, the data of the project indicated that telomere shortening can trigger HHV-6 reactivation, revealing a link between viral latency and cellular aging. These results advance our understanding of virus–host interactions but also provide tools for future research.