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.

Keywords: chromatin 3D architecture; epigenetics; gene expression; herpesvirus (hhv-6); latency.

Figure 1

RNA-Seq time course reveals the establishment of transcriptionally silent state upon human herpesvirus 6A (HHV6A) infection. (A) 293T cells were infected with ciHHV-6A and green fluorescent protein (GFP) expressing cells were isolated by FACS. RNA-seq analysis was performed on the cells during the integration phase at the indicated days post infection (dpi). (B) Cluster analysis of RNA-seq expression of HHV-6A GFP genes, showing expression values of normalized counts with a pseudo-count of one. (C) Boxplots of mean viral gene expression shown for each time point group. (D) RNA-seq signal plotted across the HHV-6A genome for each day. Viral expression is almost completely absent by day 7 indicating the establishment of a transcriptionally silent latent infection. At the later timepoints (e.g., days 4-6), two prominent peaks can be seen flanking the U41 and U42 genes, the origin of lytic DNA replication (oriLyt) and the CMV promoter and GFP regions.

Figure 2

Chromatin interactions of chromosomally integrated human herpesvirus 6A (HHV-6A). Circular Chromosome Conformation Capture (4C-seq) assay was employed using chromosomally integrated HHV-6A cell models to investigate virus-host chromatin interactions. (A) 4C-seq assay with ciHHV-6A cells. The integrated HHV-6A genome (yellow) is shown with its flanking telomeric regions (gray) and telomere-proximal unique portions of a human chromosome (black). Indicated are the restriction endonucleases sites used in the 4C-seq assay (HindIII and DpnII are indicated as ‘H’ and ‘D’, respectively). Inverse PCR was performed with distinct viewpoint primers and analysis interaction regions was performed as shown. (B) HHV-6A trans interactions for in vitro integration model (ciHHV-6A) and the chromosomally inherited iciHHV-6A cell model (iciHHV-6A). Significant interactions are visualized using circos plots both viewpoints are shown for ciHHV-6A cells (left) and for iciHHV-6A cells(right). (C) HHV-6A cis interactions identified for two separate HHV-6A viewpoints. Inverse primers were designed for two viewpoint regions chosen at ~ 64 kb and 148 kb along the HHV-6A genome. Note that the HHV6A-GFP genome is slightly larger than the canonical HHV-6A genome due to the additional presence of recombinant elements. (D) Host interaction regions (trans) are annotated using the 127 epigenomes 25-state chromatin state model database for ciHHV-6A (left) and iciHHV-6A (right). The host interaction regions contain the indicated chromatin database annotations. Displayed in boxplots (top) and as a heatmap (below).

Figure 3

(A) Identification of chromosome ends harboring integrated human herpesvirus 6A (HHV-6A). (A) Genome-wide karyotype plot showing 4C-seq read mapping density for iciHHV-6A, viewpoint 6a10 replicate 1, across each human chromosome. (B) Karyotype plots showing 4C-seq read mapping density for chromosomes 15 and 19 for each cell-line. (C) 4C-seq signal clustering method to score all the reads within 500 kb from each chromosomal end. The sum of these scores is represented in the bar plots for each replicate for each viewpoint in both ciHHV-6A and iciHHV-6A cell types. (D) Statistical measure of the likelihood that a specific chromosome harbors integrated HHV-6A. For datasets that contain replicates, an ANOVA was performed using the score from our algorithm across all chromosome ends followed by pairwise Tukey’s HSD post-hoc tests. The “p-score” is the mean -log10(p-value) value for all pairwise comparisons divided by (normalized by) the maximum mean -log10(p-value). (E) Integration sites were confirmed via fluorescent in situ hybridization using probes complimentary to chr19q (green) and the HHV-6A genome (red) in iciHHV-6A (SMC) cells.

Figure 4

MinION-based 4C-seq human herpesvirus 6A (HHV-6A) integration analysis. (A) Karyotype plots showing 4C-seq MinION read mapping density at chr15q and chr19q for ciHHV-6A and iciHHV-6A cell models. (B) Chromosomal end integration site analysis using 4C-seq with MinION reads. As in Figure 3D , a statistical measure of the likelihood that a specific chromosome harbors integrated HHV-6A. Plotted are only the top scoring chromosome ends. (C) A titration series of cell dilutions was used for 4C-seq MinION analysis and was scored with statistical testing to identify potential HHV-6A integration chromosome ends. (D) MinION 4C-seq analysis results of a frozen iciHHV-6B+ human lymphocyte sample.