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Title: The Telomeric Repeats of Human Herpesvirus 6A (HHV-6A) Are Required for Efficient Virus Integration}
Article:
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.
Introduction:
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[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. First evidence for HHV-6 integration dates back to 1993[11], but over the years both virus species were shown to integrate virtually exclusive into the telomere region of host chromosomes in latently infected cells[12–15], while no circular episomes were detected. In addition, the virus can also 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[16–24]. The actual percentage is dependent on geographic origin and demographics of the cohort and has been shown to vary from 0. 6% to 2. 7% in large cohort studies. In a recent study, the relative incidence of iciHHV-6A and iciHHV-6B was determined to be 41% and 59%, respectively[24]. HHV-6 can reactivate from latently infected cells and in iciHHV-6 patients, which in both cases is associated with several diseases including encephalitis as well as infections or graft rejection following transplantation[25, 26]. While the integration is a well-established phenomenon, the underlying molecular mechanism remains completely unknown. HHV-6 has a class A genome of about 160 kb in length and is composed of one unique segment( U) that is flanked by direct repeats( DR). The HHV-6 genome harbors two distinct arrays of telomeric repeats( TMR) at the ends of its DR region. At the right genomic terminus of the DR are the perfect TMR( pTMR), a repeat array of the hexanucleotide TTAGGG identical to the human telomere sequences. At the left end of the DR are the imperfect TMR( impTMR), telomeric repeats that are interrupted by related hexamers[27–30]. The number of TMR varies from 15 to 180 copies among clinical isolates and different laboratory strains[29]; with HHV-6B having slightly longer and more complex TMR sequences[31]. Even though TMR have been identified in the HHV-6 genome many years ago, the role of the HHV-6 TMR remained elusive. We hypothesized that the TMR could facilitate the integration of HHV-6 into the telomeres of host chromosomes by homologous recombination. In this study, we determined the role of the TMR arrays in HHV-6A replication and integration. We generated recombinant viruses that lack all TMR or only harbor either pTMR or impTMR. Analysis of their replication properties revealed that deletion of the TMR does not affect HHV-6A replication. To determine integration efficiencies of the recombinant viruses, we established an in vitro integration system that allowed us to measure the integration efficiency and genome maintenance by fluorescence in situ hybridization( FISH) and qPCR, respectively. Using this system, we could demonstrate that the TMR are essential for efficient virus integration and that the pTMR are the key component for integration, while the impTMR only play a minor role. Our study provides the first molecular evidence on the HHV-6A integration mechanism and the viral sequence elements that facilitate its integration.
Discussion:
Most herpesviruses maintain their viral genome as extra-chromosomal circular episomes during latency[31]. 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[15]; 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[30]. 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. To address the role of the HHV-6A TMR, we generated virus mutants lacking either the impTMR( ΔimpTMR), the pTMR( ΔpTMR) or both in combination( ΔTMR) using the HHV-6A BAC. The recombinant viruses replicated comparable to wt and revertant viruses, confirming that the TMR are dispensable for HHV-6A replication and indicating that the absence of the TMR has no effect on circularization of the virus. To quantify the integration efficiency of these mutants, we established an integration assay that allowed us to address this question using a pure population of infected cells. In our FISH analyses, we observed that integration occurred in various chromosomes, judging by their size and shape, but always at the end of both chromatids of metaphase chromosomes. Episomal genomes would not specifically localize to a single chromosome and the corresponding chromatids. Using this system, we could demonstrate that HHV-6A integration is severely impaired in the absence of the TMR and that the genome is rapidly lost. Despite our efforts, we were not able to generate clonal cell lines for the ΔTMR mutant virus that harbor the HHV-6A genome, supporting our model that the TMR are central for efficient integration and maintenance of the virus genome. Viruses that merely lack the pTMR, which display 100% identity with the human telomere sequences, also displayed significantly impaired integration capacity. This suggests that the pTMR could facilitate the first recombination event via homologous recombination with the host telomeres. Our conclusion is consistent with several studies obtaining sequence analyses in iciHHV-6 patients. The integration sites of HHV-6 in vivo were mapped to the pTMR of DRR being fused to the telomere/subtelomere region of the human chromosomes[15, 32–36]. The impTMR alone in the ΔpTMR mutant were not sufficient for efficient viral integration, as only very few integration events occurred. The residual integration events observed for the ΔpTMR mutant virus could be explained by a recombination between the impTMR and the host telomeres. As the impTMR are not completely homologous to the host telomeres, the recombination efficiency is likely lower than for the pTMR. In patients, this less efficient integration event has already been observed in one iciHHV-6 patient, in which the impTMR could be demonstrated as the site of integration[32]. Intriguingly, deletion of the impTMR only had a minor impact on HHV-6A integration frequencies, suggesting that the impTMR are less important for the HHV-6A integration process. If the pTMR would indeed facilitate the recombination with the telomeres, the impTMR would represent the end of the host chromosome and could serve as a starting point to extend the telomeres. In this scenario, an increased length of the impTMR as we discovered in the HHV-6A wt BAC would be beneficial for the establishment of the new telomere. Upon deletion of the impTMR, the new chromosome termini( DR of HHV-6A) could be identified as a double strand break, which in turn would induce DNA damage responses. Finally, this could result in a loss of some of the cells that harbor the integrated virus genome, unless the telomeres are restored by other means. Besides HHV-6A, already 16 other herpesviruses have been discovered that harbor TMR at the end of their genomes[31, 37]. Aside from the TMR, these viruses are very diverse, representing almost all genome classes, all Herpesviridae subfamilies and even the Alloherpesviridae that infect fish. The wide conservation of these repeats suggests that the TMR perform an important function for these herpesviruses. Our data suggest that the TMR of HHV-6A and also other viruses facilitate integration into the host telomeres, depicting a yet unappreciated mechanism for the maintenance of the virus genome in latently infected cells. Integration does not only occur upon infection in vitro, but also plays an important role in vivo. We and other groups demonstrated that the highly oncogenic alphaherpesvirus Marek’s disease virus( MDV) can also integrate its genome into the telomeres of latently infected host chicken cells[38–40] providing thereby an optimal natural virus/host small animal model for herpesvirus integration[41]. Integration of MDV plays an important role in the pathogenesis, as it is a prerequisite for lymphoma formation in the chicken[38]. In addition, integration into host telomeres not only allows stable maintenance of the virus genome in latently infected and tumor cells but also permits efficient mobilization of the virus genome during reactivation from latency[38]. Despite recent advances, more work needs to be done to fully understand the integration mechanism that allows the maintenance of the genome of HHV-6A, MDV and potentially other viruses in latently infected cells.
Herpesviruses are ubiquitous pathogens that persist in the host for life . Two human herpesviruses ( HHV-6A and HHV-6B ) can integrate their genetic material into the telomeres of host chromosomes . Integration also occurs in germ cells , resulting in individuals that harbor the virus in every single cells of their body and transmit it to their offspring , a condition that affects about 1% of the human population . We set to elucidate the integration mechanism that allows these viruses to maintain their genome in infected cells . Intriguingly , HHV-6A , HHV-6B and several other herpesviruses harbor telomere sequences at the end of their genome . Removal of these sequences in the genome of HHV-6A revealed that the viral telomeres are crucial for the integration of this human herpesvirus . In addition , we demonstrate that the telomere sequences at the right and left end of the virus genome play different roles in the integration process . Taken together , our data sheds light on the integration mechanism that allows HHV-6A to integrate into somatic cells and to enter into the germ line .