Benedikt Kaufer, Associate Professor Free University, Berlin, Germany

Surprise finding : HHV-6 telomeric repeats are crucial for HHV-6 integration

In All, ciHHV-6, Latest Scientific News, Molecular Biology by Kristin Loomis

When the research team led by Benedikt Kaufer attempted to shed light on the mechanism behind HHV-6 integration, they were suprised to find that the gene U94 was not critical to the integration process. Since the HHV-6 specific U94 gene shares homology and biological properties with the adenovirus associated virus (AAV) Rep68, the gene responsible for AAV integration into human chromosomes, it was considered the most likely candidate to mediate HHV-6 integration.

This unexpected result forced Kaufer’s team to search for other HHV-6 elements involved in integration.  They focused on the regions that are identical or nearly identical to the telomeric repeats at the extremities of human chromosomes. When these regions were removed from the HHV-6 genome, the mutant virus was severely impaired in its ability to integrate into the human chromosome. On the other hand, when U94 was removed, the virus still integrated equally well into human chromosomes.

Integration of human herpesvirus 6 into human chromosomes is still a very poorly understood biological phenomenon. Several important questions remain:

  • What conditions favor integration versus a lytic infection?
  • Is the expression of certain HHV-6 proteins necessary for integration to occur?
  • Are certain cells more susceptible to integration?

Equally important biological questions relate to the biological outcome of integrated HHV-6.  We know that treating ciHHV-6 cells with HDAC inhibitors stimulates the expression of genes. It is therefore likely that a number of prescription drugs promote HHV-6 gene expression and may cause the release of integrated virus. This could have clinical consequences, especially in patients taking immunosuppressants.  We also know that the integrated virus can activate and produce infectious virus in severely immunocompromised patients (Endo 2014).

Although our knowledge on chromosomally-integrated HHV-6 (ciHHV-6) is still in its infancy our understanding of ciHHV-6 is rapidly progressing thanks to the pioneering and collaborative efforts of multiple scientists over the past six years.

The first author on both studies was Kaufer’s doctoral student Nina Wallaschek.

More detail on the two studies can be found below :

Wallaschek 2016a

In the first study, the researchers examined the HHV-6 gene U94, which was previously thought to facilitate virus integration as U94 possesses all molecular functions required for recombination, such as ATPase, helicase, and exonuclease, as well as single-strand and double-strand DNA binding activity with a preference for TTAGGG repeats like those present in human telomeres. In order to determine the role of U94 in HHV-6 integration, the entire U94 gene (DU94) was deleted in wild-type (wt) HHV-6A. A revertant virus was also generated to ensure that no secondary mutations occurred during the mutagenesis procedure. In multi-step growth kinetics, the DU94 mutant exhibited a significant growth defect compared to the wt and revertant viruses. However, subsequent in vitro integration assays and FISH analyses revealed that DU94 integrates with a frequency comparable to wt and revertant viruses, indicating that U94 is not essential for HHV-6A integration. Furthermore, the researchers excluded a major influence of the cellular recombinase Rad51 in the integration process using the Rad51-specific inhibitor RI-1 in their setup.

Wallaschek 2016b

The second study by the same group targeted the telomeric repeats present at the ends of the HHV-6 genome. In its direct repeat regions (DR), HHV-6 harbors two distinct telomeric repeat (TMR) arrays, the perfect TMR (pTMR) and the imperfect TMR (impTMR). The pTMR at the right terminus of the DR regions are identical to the human telomere sequences. The impTMR at the left end are interrupted by related hexamers. To decipher the impact of the TMR arrays, the group generated a mutant virus termed DTMR, in which both the pTMR and impTMR were deleted. Although the DTMR mutant did not display any replication defect compared to wt virus, integration was virtually abolished. To determine the role of the individual TMR arrays, two additional mutant viruses were generated that either lack the pTMR (ΔpTMR) or the impTMR (ΔimpTMR). The DpTMR mutant showed severely impaired integration similar to the observations in the double deletion mutant. Nevertheless, there was residual integration presumably at the end of the host chromosomes accounted for by the remaining impTMRs. Intriguingly, the DimpTMR mutant only revealed a two-fold reduction in integration frequency compared to the wt virus. Taken together, these findings suggest that the pTMR play the major role in HHV-6A integration and that the impTMR aid in this process. Overall, these studies provide important insights into the mechanism that facilitates integration of HHV-6 into somatic cells and the germ line.

Kaufer Lab, Free University

Kaufer Lab, Free University