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

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.

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

“…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).…”

“…The integrated virus genome can be excised from latently infected cells and reinitiate lytic replication (5,(46)(47)(48)(49). Certain HHV-6A/B proteins, such as U94, possess many characteristics, such as exonuclease and helicase activities, a preferential DNA binding to telomeric sequences, and the ability to hydrolyze ATP, all of which are compatible with a role in viral integration (50).…”

“…Wallaschek et al recently demonstrated that the viral telomeric repeats are required for efficient HHV-6A integration into host telomeres (28). 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).…”

“…HHV-6A/B integration can occur in various chromosomes, with viral genomes consistently detected in telomeric regions that are located at chromosome termini (5,10,(13)(14)(15). Telomeres are composed of double-stranded TTAGGG repeats (8 to 13 kbp long) followed by a 30-to 200-bp single-stranded 3= overhang (16)(17)(18)(19).…”

“…During latency, human herpesviruses typically maintain their genomes as extrachromosomal nuclear episomes. How HHV-6A/B achieve latency is still unclear, and since these viruses can integrate their genomes into host chromosomes, integration is considered one possible mode of latency (5). HHV-6 integration was first described by Luppi et al, who observed the presence of the integrated HHV-6 genome in the chromosomes of freshly isolated peripheral blood mononuclear cells (PBMC) (6,7).…”