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. 2010 Mar 23;107(12):5563-8.
doi: 10.1073/pnas.0913586107. Epub 2010 Mar 8.

The latent human herpesvirus-6A genome specifically integrates in telomeres of human chromosomes in vivo and in vitro

Jesse H Arbuckle  1 , Maria M Medveczky, Janos Luka, Stephen H Hadley, Andrea Luegmayr, Dharam Ablashi, Troy C Lund, Jakub Tolar, Kenny De Meirleir, Jose G Montoya, Anthony L Komaroff, Peter F Ambros, Peter G Medveczky
Affiliations

The latent human herpesvirus-6A genome specifically integrates in telomeres of human chromosomes in vivo and in vitro

Jesse H Arbuckle et al. Proc Natl Acad Sci U S A. .

Abstract

Previous research has suggested that human herpesvirus-6 (HHV-6) may integrate into host cell chromosomes and be vertically transmitted in the germ line, but the evidence--primarily fluorescence in situ hybridization (FISH)--is indirect. We sought, first, to definitively test these two hypotheses. Peripheral blood mononuclear cells (PBMCs) were isolated from families in which several members, including at least one parent and child, had unusually high copy numbers of HHV-6 DNA per milliliter of blood. FISH confirmed that HHV-6 DNA colocalized with telomeric regions of one allele on chromosomes 17p13.3, 18q23, and 22q13.3, and that the integration site was identical among members of the same family. Integration of the HHV-6 genome into TTAGGG telomere repeats was confirmed by additional methods and sequencing of the integration site. Partial sequencing of the viral genome identified the same integrated HHV-6A strain within members of families, confirming vertical transmission of the viral genome. We next asked whether HHV-6A infection of naïve cell lines could lead to integration. Following infection of naïve Jjhan and HEK-293 cell lines by HHV-6, the virus integrated into telomeres. Reactivation of integrated HHV-6A virus from individuals' PBMCs as well as cell lines was successfully accomplished by compounds known to induce latent herpesvirus replication. Finally, no circular episomal forms were detected even by PCR. Taken together, the data suggest that HHV-6 is unique among human herpesviruses: it specifically and efficiently integrates into telomeres of chromosomes during latency rather than forming episomes, and the integrated viral genome is capable of producing virions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Vertical agarose gel technique identifies HHV-6 present in the host genomic fraction of Family-1 T cells—1 million T cells isolated from Family-1 members, control uninfected PBMCs, and HHV-6 positive Katata cell line (HHV-6B integrated Burkitt's lymphoma cell line) (37). Cells were loaded on a vertical agarose gel and analyzed for episomal, linear, or integrated DNA by the method of Gardella et al. (21). (A Left) Southern hybridization with HHV-6A probe. (A Right) Blot was stripped and hybridized with mitochondrion oligonucleotide probe (41). (B) T cells of family members immortalized with H. saimiri strain C484 were subjected to Gardella gel analysis and hybridized with HHV-6 and H. saimiri probes.
Fig. 2.
Fig. 2.
Chromosome 17p subtelomere-specific PCR from DNA of members of Family-2. (A) The putative HHV-6-chromosome 17p-HHV-6A junction. Arrows indicate position of primers (drawing is not to scale) derived from the direct repeat left (DRL) and right (DRR) of the viral genome (Unique long = UL) and to chromosome 17p subtelomere (28). (B) Genomic DNA subjected to amplification from Family-2 Mother and Sibling, HHV-6A-infected and uninfected Jjhan cells. PCR products were separated by electrophoresis and analyzed by Southern blotting using 32P-labeled oligonucleotide probes as indicated at the bottom of each panel. Cohybridization of a 1.5-kb fragment with all three probes suggests HHV-6A chromosome joining with viral DRR telomere repeat. (C) The predominant 1.5-kb amplicon from Family-2/Mother was cloned (n = 3) and sequenced. A GenBank homology search confirmed the presence of chromosome subtelomere 17p sequence (upper) joined with TTAGGG telomere repeats (bold and underlined), and HHV-6 DRR sequence (lower).
Fig. 3.
Fig. 3.
PCR amplification fails to detect HHV-6 DNA in episomal fractions of CsCl/ethidium bromide (EtBr) gradients. To search for covalently linked circular viral episomes by a method more sensitive than the method of Gardella et al., 50 μg of DNA from two latently infected HEK-293 clones, T cells from Family-2/Mother, and T cells from Family-1/Sibling-2 immortalized with HVS strain C484 were subjected to CsCl/EtBr gradient ultracentrifugation for 2 days (CsCl density 1.55 g/mL, 10 μg/mL EtBr; VTi 65 Rotor at 40,000 rpm). After centrifugation, fractions were collected, and linear and episomal (ccc) DNA was identified by agarose electrophoresis. Salt and EtBr were removed from combined linear and episomal ccc fractions and subjected to PCR based amplification using primers to HHV-6 ORF-U94 and mitochondrial cytochrome c oxidase (positive episomal control) (A), HHV-6 ORF-U94 and β-actin genomic positive control (B), and HHV-6 ORF-U94, C484 Stp, and cytochrome c oxidase (positive episomal control) (C). ccc, covalently closed circular episomal fraction.
Fig. 4.
Fig. 4.
HHV-6 DNA qPCR analysis of patient T cells and in vitro latently infected HEK-293 cell lines induced by TPA and TSA. T cell cultures from five family members and three latently infected HEK-293 cell lines were cultured in AIM-V or DMEM medium supplemented with 10% FCS and treated with known inducers of herpesvirus lytic replication protein kinase-C inducer TPA (20 ng/mL) and histone deacetylase inhibitor trichostatin-A (TSA) (80 ng/mL) for 3 days (42, 43). DNA isolated from cells (in triplicate) was subjected to quantitative real-time PCR (qPCR) for ORF U94 and fold-change ratios of Ct values normalized to β-actin were relative to untreated control. (A) HEK-293 cells (n = 3) (Fig. S8). (B) T cells (n = 5) (Fig. S8). TSA promoted a significant increase in viral DNA replication, whereas the stimulation with TPA and hydrocortisone had a milder effect. Statistical analysis was based on the Student t test, and P < 0.05 was considered significant. (C) Gardella gel analysis of infected Molt-3 cells 2 weeks after coculturing with PBMCs treated with TPA from Family-1/Sibling-2—Hector 2 T cell line carrying one copy of HHV-6A per a cell.
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