Date Published: May 31, 2016
Publisher: Public Library of Science
Author(s): Nina Wallaschek, Anirban Sanyal, Fabian Pirzer, Annie Gravel, Yasuko Mori, Louis Flamand, Benedikt B. Kaufer, Paul D. Ling.
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.
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 [1–4]. Primary infection with HHV-6B occurs during early childhood until the age of two [5–7]. HHV-6B is the most common causative agent of the febrile illness roseola infantum (sixth disease) and in rare cases causes severe neurological complications such as seizures and encephalitis [8, 9]. Greater than 90% of the adult human population is seropositive for HHV-6B. The epidemiology and disease association with HHV-6A, which was initially discovered in patients with lymphoproliferative disorders , is much less characterized.
Most herpesviruses maintain their viral genome as extra-chromosomal circular episomes during latency . In the case of HHV-6, Arbuckle and colleagues found no evidence for viral episomes in latently infected cells in vitro, but instead detected the integrated virus genome in the telomeres of host chromosomes ; however, the exact in vivo latency reservoir of HHV-6 is still poorly understood. Several groups proposed that the TMR present in the HHV-6 genome could facilitate integration . This hypothesis was mainly based on sequence analyses of the integration sites in iciHHV-6 patients, as the pTMR at the right end of the viral genome were shown to be directly fused to the telomere/subtelomere sequences of the host chromosomes [15, 32–36]. However, no experimental evidence was available that the TMR are indeed involved in this process.