Abstract
Human herpesvirus 6A (HHV-6A) and 6B (HHV-6B) are ubiquitous betaherpesviruses that infects humans within the first years of life and establishes latency in various cell types. Both viruses can integrate their genomes into telomeres of host chromosomes in latently infected cells. The molecular mechanism of viral integration remains elusive. Intriguingly, HHV-6A, HHV-6B and several other herpesviruses harbor arrays of telomeric repeats (TMR) identical to human telomere sequences at the ends of their genomes. The HHV-6A and HHV-6B genomes harbor two TMR arrays, the perfect TMR (pTMR) and the imperfect TMR (impTMR). To determine if the TMR are involved in virus integration, we deleted both pTMR and impTMR in the HHV-6A genome. Upon reconstitution, the TMR mutant virus replicated comparable to wild type (wt) virus, indicating that the TMR are not essential for HHV-6A replication. To assess the integration properties of the recombinant viruses, we established an in vitro integration system that allows assessment of integration efficiency and genome maintenance in latently infected cells. Integration of HHV-6A was severely impaired in the absence of the TMR and the virus genome was lost rapidly, suggesting that integration is crucial for the maintenance of the virus genome. Individual deletion of the pTMR and impTMR revealed that the pTMR play the major role in HHV-6A integration, whereas the impTMR only make a minor contribution, allowing us to establish a model for HHV-6A integration. Taken together, our data shows that the HHV-6A TMR are dispensable for virus replication, but are crucial for integration and maintenance of the virus genome in latently infected cells.
Highlights
In 2012, the two previously described variants Human herpesvirus 6A (HHV-6A) and HHV-6B were classified as separate virus species based on differences regarding their genetic and biological characteristics including variations in DNA sequences, distinct restriction patterns and specific reactivity to monoclonal antibodies [1,2,3,4]
Southern blotting, using a specific probe for the telomeric repeats (TMR) (Fig 1C). Both wild type and ΔTMR mutant were reconstituted in JJHan cells by nucleofection of BAC DNA
Replication kinetics in JJHan cells revealed that replication was comparable between wt and ΔTMR mutant virus (Fig 1D and S1A Fig), confirming that the TMR are dispensable for HHV-6A replication in JJHan cells
Summary
In 2012, the two previously described variants HHV-6A and HHV-6B were classified as separate virus species based on differences regarding their genetic and biological characteristics including variations in DNA sequences (especially in the IE region), distinct restriction patterns and specific reactivity to monoclonal antibodies [1,2,3,4]. The epidemiology and disease association with HHV-6A, which was initially discovered in patients with lymphoproliferative disorders [10], is much less characterized Upon primary infection, both HHV-6A and HHV-6B (HHV-6) establishes latency, which allows the virus to persist in the host for life. The virus can integrate into germ cells, resulting in vertical transmission and individuals that harbor the integrated virus in every single cell of their body This condition is termed inherited chromosomally integrated HHV-6 (iciHHV-6) and is present in about 1% of the human population [16,17,18,19,20,21,22,23,24]. While the integration is a well-established phenomenon, the underlying molecular mechanism remains completely unknown
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