Fig 1.
Sampling of HTLV-1 infected cells along expression cycle ex vivo.
(A) Overview of sample preparation and collection. (B) Gating scheme used to obtain single live lymphocytes from peripheral blood lymphocyte samples. (C) Gating of CADM1high cells was performed based on CADM1 levels in uninfected controls. Dotted line represents CADM1 intensity threshold for infected samples. (D) ddPCR measurement of PVL of CADM1high-sorted cells used for RNA-seq at t0. Infected T cell clone 11.50, which contains a single copy HTLV-1 provirus per cell [34,112], was used as a positive control. Columns and error bars represent mean ±SEM.
Fig 2.
RNA-seq dataset captures progression of infected cells along HTLV-1 expression stages.
(A) Trajectory of sequenced reads mapped to the HTLV-1 sense-strand, excluding the LTRs. (B) Correlation between RNA-seq and qRT-PCR HTLV-1 transcript trajectories. Dotted line represents linear model fit. Shaded area represents linear model 95% confidence interval. Statistics shown were calculated using a paired Spearman rank correlation test. (C) Above: Schematic of proviral sequence with tax/rex and gag-pro-pol polytranscript shown. Dotted lines represent introns, boxes represent ORFs, and lines represent untranslated regions. Shaded regions correspond to AB513134 regions 804–1,433 and 7,400–8,029, used to quantify reads aligning to gag and tax regions, respectively. Below: Ratio of reads aligning to proviral tax and gag regions, corresponding to the ratio of spliced to unspliced transcripts. Summary line and error bars represent mean ±SEM. (D) Scree plot showing contribution of principal components to total variance observed, with red line representing cumulative sum. Zoom inset focuses on principal components accounting for ≈ 80% of variance in the data. (E) PCA bi-plot of infected and uninfected cell gene expression.
Fig 3.
Timepoints separated by distinct changes in gene expression specific to infected cells.
(A) Heatmap of genes differentially expressed over time between infected and uninfected samples. (B) Top five (based on FDR-corrected p value) non-synonymous gene ontology (GO) biological process terms associated with each interval. (C) Summary of mean-normalised trajectories of genes for GO terms presented in panel B. Discontinuous vertical lines indicate sampling timepoints. Shaded areas represent ±SEM.
Fig 4.
HTLV-1 infection does not alter responsiveness to reactivating factors.
(A) ORA using Hallmark gene sets of genes differentially expressed in uninfected samples during their introduction to ex vivo culture (0–24 hrs.). (B) log2-fold changes during reactivation (0–24 hrs.) in infected and uninfected samples. BH FDR-corrected paired Wilcoxon test p values shown. (C) ORA of genes differentially expressed between infected and uninfected samples at 0 hours. (D) Differential expression of genes within Hallmark sets between infected and uninfected samples at 0 hours. Summary bars represent mean ±SEM. (E–F) Trajectories of key TNFα signaling via NF-κB, and mTORC1 pathway components. Asterisks indicate genes which are significantly differentially expressed between infected and uninfected samples at 0 hours (Wald test; BH FDR-corrected p < 0.05). Summary lines and shaded areas represent mean ±SEM. Insets highlight factors with positive (blue) or inhibitory (red) effect on pathway.
Fig 5.
Polycomb effectors are differentially expressed during reactivation.
(A) Mean trajectories of genes significantly (paired Spearman rank correlation test; FDR-corrected p < 0.05) correlated with sense-strand transcript levels. Shading represents ±SEM. (B) Top 10 terms ranked by p-value. (C) Fraction of genes annotated as histone ubiquitination-associated correlated either positively or negatively with sense-strand expression. (D) Fraction of genes annotated as histone ubiquitination-associated differentially expressed over time between infected and uninfected samples (Likelihood ratio test; BH FDR-corrected p < 0.05). (E) Expression trajectories of PRC1 complex components. Asterisks denote significant differential expression between infected and uninfected samples (Likelihood ratio test; BH FDR-corrected p < 0.05). Coloured boxes represent PRC complexes comprising each gene [30]. Shaded areas represent ±SEM. (F) Trajectories of PRC2 components. (G) Trajectories of PR-DUB complex components and associated factors.
Fig 6.
Knockdown of BAP1, OTUD5 or USP14 reduces proviral transcript levels in infected cells.
(A) Trajectories of PR-619 targets in infected and uninfected cells. Asterisks denote genes significantly differentially expressed in infected samples between 0–24 hours (Wald test; BH FDR-adjusted p < 0.05). Summary lines and shaded area represent mean ±SEM. (B) Knockdown efficiency following incubation with siRNA. Dot colours represent individual infected T cell clones carrying distinct proviral integration sites. Error bars ±SEM. (C) Viability measurements at 72 and 96 hours following the addition of siRNA targeting DUBs. Error bars ±SEM. (D) Changes in transcript levels upon DUB inhibition after 72 and 96 hours. Error bars ±SEM.
Table 1.
HTLV-1+ subject and uninfected volunteer samples used.
Table 2.
ddPCR primer and probe sequences [33].
Table 3.
Thermal cycler programme used for ddPCR.
Table 4.
Thermal cycler protocol for qPCR.
Table 5.
Primers used for qRT-PCR [29].
Table 6.
Channels used in acquisition of smFISH images.
Table 7.
Details of clonal HTLV-1-infected T cells used.
Table 8.
Primers used to examine candidate deubiquitinase expression levels.
Primers were designed against the "Ensembl Canonical" transcript variant for each gene. Annealing sites matching exon-exon splicing points were selected.