Flow sorting of provirus-expressing and non-expressing cells by an independent technique produced similar q4C profiles.

(A) q4C profiles of Tax−(upper panel) and Tax+ (lower panel) cells sorted from clone TBX4B after intracellular staining of Tax. (B) q4C profile of non-expressing (GFP-) (upper panel) and provirus-expressing (GFP+) (lower panel) cells isolated from d2EGFP-TBX4B clones, selected by GFP signal (without Tax staining). (TIFF)</p

DNA sequence of primers and probes used in 3C-qPCR and RT-qPCR.

DNA sequence of primers and probes used in 3C-qPCR and RT-qPCR.</p

Treatment with flavopiridol for 1.5 hrs did not alter Tax protein expression.

Clone 11.63 cells were treated with 1nM flavopiridol for 1.5 hrs and then stained for Live/Dead and then Tax protein. (TIFF)</p

Expression of distant host genes correlates with expression of <i>tax</i>.

(A) Normalized mRNA read counts of two genes that lie >1.4 Mb from the provirus in clone Timer-TBX4B, in the four successive phases of the HTLV-1 plus-strand transcriptional burst: DN–double negative (HTLV-1 silent); blue–early burst; DP double-positive (mid-burst); red–late burst. Results of two independent experiments are shown. Expression of both SMC1B and RIBC2 closely followed the trajectory of the HTLV-1 burst in clone Timer-TBX4B, but not in the unrelated HTLV-1-infected clone Timer-3.60. Data from [11]. (B) Knockout of the CTCF binding site in the provirus in clone Timer-TBX4B (middle panel) abolished the transcription of both SMC1B and RIBC2 observed in the wild-type clone (lower panel). Results of two independent experiments are shown. Neither gene was expressed in an unrelated HTLV-1-infected clone ED. The results suggest that maintenance of a CTCF-dependent chromatin loop between the host genome and the provirus is required for the burst of transcription of these distant genes associated with the HTLV-1 plus-strand burst. (TIFF)</p

Proviral and host transcription and splicing in live-sorted T cell clones.

(A) RNA-seq analysis of HTLV-1 proviral expression in live-sorted d2EGFP clones (TBX4B, 11.50 and 11.63). (B) Tax expression measured by qPCR with primers specific for tax mRNA or 18S ribosomal RNA (18S rRNA). qPCR plots are expression values normalized to 18S rRNA. Data represent a mean of two biological replicates; error bars are SEM. AU—arbitrary units. (C) Host RNA expression 30kb on either side of the proviral integration site. On the horizontal axis, positive values denote positions extending from the 3′ LTR side of the provirus; negative values denote positions 5′ of the 5′LTR. Each row shows the transcription density (normalized RNA-seq read count) flanking that genomic position in the clone indicated at the right-hand side. In each case, transcription orientation and positions are shown relative to the integrated provirus. Read density shown in blue shows transcription in the same orientation as the proviral plus-strand (same sense); red shows transcription in the opposite sense to the proviral plus-strand (antisense). (D) Identification of splice sites of viral-host fusion transcripts in d2EGFP-TBX4B clone cells. Coverage tracks of same sense transcription (blue) and antisense transcription (red) in Integrative Genomics Viewer (IGV). Exons of PNPLA3 in the 3′ side of the integration site are highlighted in yellow. (E) Fusion transcripts between an HTLV-1 plus-strand major splice donor (red, proviral exon H1 or H2) and the canonical splice acceptor site in the host PNPLA3 gene (blue, PNPLA3 exon 3) were identified in GFP+ (HTLV-1 plus-strand-expressing) cells. To identify splice sites of fusion transcripts, reads were aligned to a reference genome (hg19) containing the HTLV-1 provirus (AB513134) genome in the TBX4B clone integration site at chr22:44323198. Fusion transcripts are shown with fused sequences.</p

Flow sorting of Tax⁻and Tax⁺ cells.

HTLV-1-infected T cell clone 11.63 was stained for live cells, crosslinked in 1% formaldehyde, stained intracellularly for Tax protein, and flow-sorted to isolate Tax−and Tax+ subsets (see Methods). (TIFF)</p

Quantitative 3C (3C-qPCR) analysis confirmed decreased frequencies of chromatin looping.

(A) q4C profile of Tax−cells of clone 11.63. The technical peak seen in the q4C viewpoint (VP) and two of the main peaks (Peak 1) and (Peak 2) identified in the output of the Tax−fraction of clone 11.63. (B) As control, the frequency of chromatin interactions was quantified by 3C-qPCR on sorted Tax−and Tax+ cells, using a primer pair and Taqman probe to detect the contacts between two regions: VP and another region in the provirus (S1 Table). There was no significant difference between Tax+ and Tax−cells (combined p value = 0.932, Fisher’s method of combining p values). (C and D) Primer pairs and probe were used to detect long-range chromatin contacts between the provirus and host genome region at Peak 1 (C) or Peak 2 (D). Results of 3C-qPCR of two biological replicates (rep) are shown. Peak1 contact frequency was significantly higher in Tax−cells than in Tax+ cells (combined p value 0.012, Fisher’s method). Peak2 contact frequency was significantly higher in Tax- cells than in Tax+ cells (combined p value 0.000607, Fisher’s method). (TIFF)</p

RNA-seq analysis of HTLV-1 proviral expression in live-sorted cells of clone Timer-TBX4B.

Cells were sorted into four populations based on the fluorescence of the Timer protein, DN–double negative (HTLV-1 silent); blue–early burst; DP double-positive (mid-burst); red–late burst. [11].(A) Coverage tracks in IGV of plus strand HTLV-1 provirus transcription and (B) host gene PNPLA3 transcription. (C) Transcription in PNPLA3 exons 1 and 2 (note range on vertical axis 0 to 500). (TIFF)</p

Clone list.

A typical HTLV-1-infected individual carries >104 different HTLV-1-infected T cell clones, each with a single-copy provirus integrated in a unique genomic site. We previously showed that the HTLV-1 provirus causes aberrant transcription in the flanking host genome and, by binding the chromatin architectural protein CTCF, forms abnormal chromatin loops with the host genome. However, it remained unknown whether these effects were exerted simply by the presence of the provirus or were induced by its transcription. To answer this question, we sorted HTLV-1-infected T-cell clones into cells positive or negative for proviral plus-strand expression, and then quantified host and provirus transcription using RNA-seq, and chromatin looping using quantitative chromosome conformation capture (q4C), in each cell population. We found that proviral plus-strand transcription induces aberrant transcription and splicing in the flanking genome but suppresses aberrant chromatin loop formation with the nearby host chromatin. Reducing provirus-induced host transcription with an inhibitor of transcriptional elongation allows recovery of chromatin loops in the plus-strand-expressing population. We conclude that aberrant host transcription induced by proviral expression causes temporary, reversible disruption of chromatin looping in the vicinity of the provirus.</div

Treatment of T cell clones with a transcriptional elongation inhibitor allows recovery of chromatin loop formation in the Tax⁺ population.

(A) After treatment with 1 nM flavopiridol (FP) for 1.5 hrs, total RNA was extracted from clone 11.63 and subjected to RT-qPCR for tax and three regions in the 3′ flanking host genome, respectively a: +188 bp, b: +535 bp and c: +3,198 bp from the 3′ end of the provirus. (B) Relative expression intensity (normalized to 18s rRNA) of tax and the host genome at positions a, b and c. Data are mean ± SEM. (N = 3). * P+ cells (middle track), and flavopiridol (FP)-treated Tax+ cells (bottom track). Diamonds mark the positions of reproducible chromatin contact sites identified by the peak calling algorithm. Open arrowheads denote positions of CTCF-BS; the filled arrowhead denotes the CTCF-BS in the provirus. Gene track shows RefSeq protein-coding genes in the flanking host genome.</p