Graduation Year

2011

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Medical Sciences

Major Professor

Peter G. Medveczky, M.D.

Committee Member

Robert J. Deschenes, Ph.D.

Committee Member

Andreas Seyfang, Ph.D.

Committee Member

Alberto van Olphen, Ph.D., D.V.M.

Keywords

Human Herpesvirus 6, HHV-6, viral latency, genome integration, telomere, chromosome, germ-line transmission, recombination, viral reactivation, integrase, central nervous system diseases

Abstract

While the latent genome of most Herpesviruses persists as a nuclear circular episome, previous research has suggested that Human Herpesvirus 6 (HHV-6) may integrate into host cell chromosomes, and be vertically transmitted in the germ-line. Because the HHV-6 genome encodes a perfect TTAGGG telomere repeat array at the right end direct repeat (DRR) and an imperfect TTAGGG repeat at the end of the left end direct repeat (DRL), we established a hypothesis that during latency, the HHV-6A and HHV-6B genome integrates into the telomeres of human chromosomes through homologous recombination with the n(TTAGGG) viral repeats, and the integrated virus can be induced to lytic replication.

We sought, first, to definitively illustrate the in vitro and in vivo integration of HHV-6A and HHV-6B. Following infection of naïve Jjhan and HEK-293 cell lines by HHV-6A and Molt3 cell line by HHV-6B, the virus integrated into telomere of chromosomes. Next, 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 ml of blood. FISH confirmed that HHV-6 DNA co-localized with telomeric regions of one allele on chromosomes 17p13.3, 18q23, and 22q13.3, while the integration site was identical among members of the same family. Partial sequencing of the viral genome identified the same integrated HHV-6A strain within members of families, confirming vertical transmission of the viral genome through the germ-line [inherited HHV-6 (iHHV-6)].

Amplification and sequencing of the HHV-6A and more recently HHV-6B viral-chromosome junction identified DRR integrated into the telomere directly adjacent to the subtelomere of the chromosome. After mapping the DRR of iHHV-6, we subsequently focused on determining if the DRL was present in the integrated genome and whether the remaining telomere sequence of the chromosome was extended beyond the DRL. Southern hybridization of PCR amplified HHV-6 integrated cell lines and iHHV-6 patients PBMCs indicate the presence of DRL within the integrated viral genome. Therefore, the genomic structure of the iHHV-6 is as follows: chromosome-subtelomere-(TTAGGG)5-41-DRR-U-DRL-(TTAGGG)n.

During latent integration, no circular episomes were detected even by PCR. However, trichostatin-A treatment of PBMCs and in vitro integrated HEK-293 cells induced the reactivation of iHHV-6 from its latent integrated state. We demonstrated the induction of integrated iHHV-6 with trichostatin-A lead to the excision of the integrated genome and generation of the U-DR-U junction which signifies circularization and/or concatemer formation of the viral genome through rolling-circle replication. Taken together, the data suggests that HHV-6A and HHV-6B are unique among human herpesviruses: they specifically and efficiently integrate into telomeres of chromosomes during latency rather than forming episomes, and the integrated viral genome is capable of producing virions.

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