Higher-Order Chromatin Structures of Chromosomally Integrated HHV-6A Predict Integration Sites

Abstract

Human herpesvirus -6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes. Viral integration can occur in several cell types, including germinal cells, resulting in individuals that harbor the viral genome in every cell of their body. The integrated genome is efficiently silenced but can sporadically reactivate resulting in various clinical symptoms. To date, the integration mechanism and the subsequent silencing of HHV-6A/B genes remains poorly understood. Here we investigate the genome-wide chromatin contacts of the integrated HHV-6A in latently-infected cells. We show that HHV-6A becomes transcriptionally silent upon infection of these cells over the course of seven days. In addition, we established an HHV-6–specific 4C-seq approach, revealing that the HHV-6A 3D interactome is associated with quiescent chromatin states in cells harboring integrated virus. Furthermore, we observed that the majority of virus chromatin interactions occur toward the distal ends of specific human chromosomes. Exploiting this finding, we established a 4C-seq method that accurately detects the chromosomal integration sites. We further implement long-read minION sequencing in the 4C-seq assay and developed a method to identify HHV-6A/B integration sites in clinical samples.

Excision of Integrated Human Herpesvirus 6A Genomes Using CRISPR/Cas9 Technology

“…To establish the removal of the integrated HHV-6A genome with the CRISPR/Cas9 system, we first used a well-characterized 293T cell line harboring the integrated virus genome (293T-6A) ( 54 , 55 ). These cells were previously infected with HHV-6A expressing green fluorescent protein (GFP) under the control of the major immediate early human cytomegalovirus (HCMV) promoter and harbor two copies of the integrated virus genome.…”

Evasion of the Host Immune Response by Betaherpesviruses

“…Chromosomal integration is a highly regulated event that is important to the establishment of betaherpesvirus latency [ 4 ]. HHV-6A and HHV-6B are known to integrate their genomes into the telomeres of human chromosomes in several different cell types, including germinal cells [ 171 ]. The HHV-6A genome in particular is transcriptionally silent after chromosomal integration.…”

“…In human 293T cells infected with HHV-6A, viral transcription is progressively silenced and is nearly undetectable by seven days post infection. The chromosomally integrated HHV-6A genome exists as condensed heterochromatin [ 172 ] and appears to reside in a compartment similar to a topologically associated domain (TAD), which is characterized by a high number of local intrachromosomal interactions [ 171 ]. A recent study examined higher-order chromatin interactions involving chromosomally integrated HHV-6A using circular chromosome conformation capture assays (4C-seq) specific to HHV-6A.…”

“…The 4C-seq assays found that most virus-host chromatin interactions were with heterochromatin, indicating that integrated HHV-6A is able to interact with repressed chromatin. These interactions may play a role in silencing viral gene expression in cells with chromosomally integrated HHV-6A [ 171 ].…”

“…As mentioned earlier, HHV-6A/B can integrate into germinal cells [ 171 , 173 ], allowing HHV-6A/B to be transferred vertically. This vertical DNA transfer results in a phenomenon known as inherited chromosomally-integrated HHV-6 (iciHHV-6), where every nucleated cell contains the integrated HHV-6 genome [ 173 ].…”