Human telomeres that carry an integrated copy of human herpesvirus 6 are often short and unstable, facilitating release of the viral genome from the chromosome

Huang, Yan; Hidalgo‐Bravo, Alberto; Zhang, Enjie; Cotton, Victoria E.; Méndez-Bermúdez, Aarón; Wig, Gunjan; Medina-Calzada, Zahara; Neumann, Rita; Jeffreys, Alec J.; Winney, Bruce; Wilson, James F.; Clark, Duncan A.; Dyer, Martin J. S.; Royle, Nicola J.

doi:10.1093/nar/gkt840

Cited by 73 publications

(170 citation statements)

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“…Even though the formal integration mechanism has yet to be demonstrated experimentally, sequencing of ciHHV-6A/B indicates that the TMR of the DR R of the genome is fused to the host chromosome telomeric repeats with loss of pac2 sequence from DR R . Such a structure is compatible with HR-mediated viral integration (5,30,31).…”

Section: Discussionmentioning

confidence: 97%

“…Analysis of the integrated virus genome revealed that the perfect TMR of the DR R region are fused to the host chromosome accompanied by a loss of the pac2 sequence, consistent with an integration mediated by HR between the viral TMR and the host telomere (5). At the other end of the viral genome, the pac1 region is also lost and additional telomeric repeats are added, suggesting that the TMR in DR L is a substrate for the generation of a new telomere (29)(30)(31).…”

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confidence: 78%

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Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

Gilbert-Girard

Gravel

Artusi

et al. 2017

J Virol

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Human herpesviruses 6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes using a mechanism that remains poorly understood. To achieve a better understanding of the HHV-6A/B integration mechanism, we made use of BRACO-19, a compound that stabilizes G-quadruplex secondary structures and prevents telomere elongation by the telomerase complex. First, we analyzed the folding of telomeric sequences into G-quadruplex structures and their binding to BRACO-19 using G-quadruplex-specific antibodies and surface plasmon resonance. Circular dichroism studies indicate that BRACO-19 modifies the conformation and greatly stabilizes the G-quadruplexes formed in G-rich telomeric DNA. Subsequently we assessed the effects of BRACO-19 on the HHV-6A initial phase of infection. Our results indicate that BRACO-19 does not affect entry of HHV-6A DNA into cells. We next investigated if stabilization of G-quadruplexes by BRACO-19 affected HHV-6A's ability to integrate its genome into host chromosomes. Incubation of telomerase-expressing cells with BRACO-19, such as HeLa and MCF-7, caused a significant reduction in the HHV-6A integration frequency (P Ͻ 0.002); in contrast, BRACO-19 had no effect on HHV-6 integration frequency in U2OS cells that lack telomerase activity and elongate their telomeres through alternative lengthening mechanisms. Our data suggest that the fluidity of telomeres is important for efficient chromosomal integration of HHV-6A and that interference with telomerase activity negatively affects the generation of cellular clones containing integrated HHV-6A.IMPORTANCE HHV-6A/B can integrate their genomes into the telomeres of infected cells. Telomeres consist of repeated hexanucleotides (TTAGGG) of various lengths (up to several kilobases) and end with a single-stranded 3= extension. To avoid recognition and induce a DNA damage response, the single-stranded overhang folds back on itself and forms a telomeric loop (T-loop) or adopts a tertiary structure, referred to as a G-quadruplex. In the current study, we have examined the effects of a G-quadruplex binding and stabilizing agent, BRACO-19, on HHV-6A chromosomal integration. By stabilizing G-quadruplex structures, BRACO-19 affects the ability of the telomerase complex to elongate telomeres. Our results indicate that BRACO-19 reduces the number of clones harboring integrated HHV-6A. This study is the first of its kind and suggests that telomerase activity is essential to restore a functional telomere of adequate length following HHV-6A integration.

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Section: Discussionmentioning

confidence: 97%

mentioning

confidence: 78%

Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

Gilbert-Girard

Gravel

Artusi

et al. 2017

J Virol

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“…The mechanisms by which HHV-6A and HHV-6B integrate into the telomeric/subtelomeric junction of host chromosomes remain unclear. Analysis of the viral genome of iciHHV-6A ϩ /B ϩ individuals indicates that all HHV-6A/B open reading frames are largely intact, with no gross rearrangements (17). However, the pac1 sequence from DR L and the pac2 sequence from DR R are lost during the integration process (17).…”

Section: Discussionmentioning

confidence: 99%

“…Analysis of the viral genome of iciHHV-6A ϩ /B ϩ individuals indicates that all HHV-6A/B open reading frames are largely intact, with no gross rearrangements (17). However, the pac1 sequence from DR L and the pac2 sequence from DR R are lost during the integration process (17). This is unlikely to have major impacts on the ability of HHV-6A/B to generate infectious virions, considering that invasion of the telomeric 3= overhang into the internal HHV-6A/B DR R -T1 could facilitate the release of a complete viral genome from the chromosome with a single reconstituted DR (17).…”

Section: Discussionmentioning

confidence: 99%

Cell Culture Systems To Study Human Herpesvirus 6A/B Chromosomal Integration

Gravel¹,

Dubuc²,

Wallaschek

et al. 2017

J Virol

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Human herpesviruses 6A/B (HHV-6A/B) can integrate their viral genomes in the telomeres of human chromosomes. The viral and cellular factors contributing to HHV-6A/B integration remain largely unknown, mostly due to the lack of efficient and reproducible cell culture models to study HHV-6A/B integration. In this study, we characterized the HHV-6A/B integration efficiencies in several human cell lines using two different approaches. First, after a short-term infection (5 h), cells were processed for single-cell cloning and analyzed for chromosomally integrated HHV-6A/B (ciHHV-6A/B). Second, cells were infected with HHV-6A/B and allowed to grow in bulk for 4 weeks or longer and then analyzed for the presence of ciHHV-6. Using quantitative PCR (qPCR), droplet digital PCR, and fluorescent in situ hybridization, we could demonstrate that HHV-6A/B integrated in most human cell lines tested, including telomerase-positive (HeLa, MCF-7, HCT-116, and HEK293T) and telomerase-negative cell lines (U2OS and GM847). Our results also indicate that inhibition of DNA replication, using phosphonoacetic acid, did not affect HHV-6A/B integration. Certain clones harboring ciHHV-6A/B spontaneously express viral genes and proteins. Treatment of cells with phorbol ester or histone deacetylase inhibitors triggered the expression of many viral genes, including U39, U90, and U100, without the production of infectious virus, suggesting that the tested stimuli were not sufficient to trigger full reactivation. In summary, both integration models yielded comparable results and should enable the identification of viral and cellular factors contributing to HHV-6A/B integration and the screening of drugs influencing viral gene expression, as well as the release of infectious HHV-6A/B from the integrated state.IMPORTANCE The analysis and understanding of HHV-6A/B genome integration into host DNA is currently limited due to the lack of reproducible and efficient viral integration systems. In the present study, we describe two quantitative cell culture viral integration systems. These systems can be used to define cellular and viral factors that play a role in HHV-6A/B integration. Furthermore, these systems will allow us to decipher the conditions resulting in virus gene expression and excision of the integrated viral genome resulting in reactivation.KEYWORDS chromosomal integration, ddPCR, telomere

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“…HHV-6 integration into human chromosomes may drive human disease via mechanisms such as chromosomal instability or epigenetic effects. 24 Telomeres carrying integrated HHV-6 may be unstable, leading to deletions, 25 and telomere length has important implications for chromosomal stability and cell viability. 26 Indeed, disruption in telomeres is associated with conditions ranging from pulmonary to hematologic diseases.…”

Section: Discussionmentioning

confidence: 99%

Outcomes of hematopoietic cell transplantation using donors or recipients with inherited chromosomally integrated HHV-6

Hill

Magaret

Hall-Sedlak³

et al. 2017

Blood

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• Inherited ciHHV-6 was detected in 1.4% of HCT recipients and 0.9% of their donors.• Acute GVHD grades 2-4 and cytomegalovirus viremia were more frequent when recipients or donors had inherited ciHHV-6.Human herpesvirus 6 (HHV-6) species have a unique ability to integrate into chromosomal telomeres. Mendelian inheritance via gametocyte integration results in HHV-6 in every nucleated cell. The epidemiology and clinical effect of inherited chromosomally integrated HHV-6 (iciHHV-6) in hematopoietic cell transplant (HCT) recipients is unclear.We identified 4319 HCT donor-recipient pairs (8638 subjects) who received an allogeneic HCT and had archived pre-HCT peripheral blood mononuclear cell samples. We screened these samples for iciHHV-6 and compared characteristics of HCT recipients and donors with iciHHV-6 with those of recipients and donors without iciHHV-6, respectively. We calculated Kaplan-Meier probability estimates and Cox proportional hazards models for post-HCT outcomes based on recipient and donor iciHHV-6 status. We identified 60 HCT recipients (1.4%) and 40 donors (0.9%) with iciHHV-6; both recipient and donor harbored iciHHV-6 in 13 HCTs. Thus, there were 87 HCTs (2%) in which the recipient, donor, or both harbored iciHHV-6. Acute graft-versus-host disease (GVHD) grades 2-4 was more frequent when recipients or donors had iciHHV-6 (adjusted hazard ratios, 1.7-1.9; P 5 .004-.001). Cytomegalovirus viremia (any and high-level) was more frequent among recipients with iciHHV-6 (adjusted HRs, 1.7-3.1; P 5 .001-.040). Inherited ciHHV-6 status did not significantly affect risk for chronic GVHD, hematopoietic cell engraftment, overall mortality, or nonrelapse mortality. Screening for iciHHV-6 could guide donor selection and post-HCT risk stratification and treatment. Further study is needed to replicate these findings and identify potential mechanisms. (Blood. 2017;130(8):1062-1069

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Human telomeres that carry an integrated copy of human herpesvirus 6 are often short and unstable, facilitating release of the viral genome from the chromosome

Cited by 73 publications

References 50 publications

Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

Cell Culture Systems To Study Human Herpesvirus 6A/B Chromosomal Integration

Outcomes of hematopoietic cell transplantation using donors or recipients with inherited chromosomally integrated HHV-6

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