Inherited chromosomally integrated human herpesvirus 6 as a predisposing risk factor for the development of angina pectoris

Gravel, Annie; Dubuc, Isabelle; Morissette, Guillaume; Sedlak, Ruth Hall; Jerome, Keith R.; Flamand, Louis

doi:10.1073/pnas.1502741112

Cited by 90 publications

(111 citation statements)

References 58 publications

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“…Four microliters (approximately 50 ng) of the DNA was mixed with primers and probes in a final volume of 10 l, mixed with an equal volume of 2ϫ Rotor-Gene multiplex PCR kit (Qiagen), and subjected to 40 cycles of amplification. The conditions for detection of HHV-6A/B were described previously (6,35). Primers and probe used are listed in Table 4.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

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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“…At that point in time, cells were detached using trypsin and DNA was isolated and tested by ddPCR for the presence of HHV-6 and the cellular reference gene RPP30, as described previously (82). U65-U66-specific primers were validated previously (12,83) and used for the detection of the integrated HHV-6 genome.…”

Section: Methodsmentioning

confidence: 99%

“…This condition is termed inherited chromosomally integrated HHV-6 (iciHHV-6) and is present in about 0.2% to 1% of the human population across the world (reviewed in references 10 and 11). In 2015, Gravel et al analyzed DNA samples from 20,000 Quebecers (Canada) for iciHHV-6 and concluded that iciHHV-6A/B represents a predisposing risk factor for angina pectoris (12).…”

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confidence: 99%

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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“…The effect of iciHHV-6 on human disease is largely unknown, given the challenge of studying relatively rare conditions. One large registry study of 19 597 adults found that angina pectoris occurred 3 times more frequently among individuals with iciHHV-6, 10 perhaps through telomeric disruption or viral gene expression stimulating chronic inflammation.…”

Section: Introductionmentioning

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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Inherited chromosomally integrated human herpesvirus 6 as a predisposing risk factor for the development of angina pectoris

Cited by 90 publications

References 58 publications

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

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

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

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

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