Dynamics and consequences of the HTLV-1 proviral plus-strand burst

Abstract

Expression of the transcriptional transactivator protein Tax, encoded on the proviral plus-strand of human T-cell leukaemia virus type 1 (HTLV-1), is crucial for the replication of the virus, but Tax-expressing cells are rarely detected in fresh blood ex vivo. The dynamics and consequences of the proviral plus-strand transcriptional burst remain insufficiently characterised. We combined time-lapse live-cell imaging, single-cell tracking and mathematical modelling to study the dynamics of Tax expression at single-cell resolution in two naturally-infected, non-malignant T-cell clones transduced with a short-lived enhanced green fluorescent protein (d2EGFP) Tax reporter system. Five different patterns of Tax expression were observed during the 30-hour observation period; the distribution of these patterns differed between the two clones. The mean duration of Tax expression in the two clones was 94 and 417 hours respectively, estimated from mathematical modelling of the experimental data. Tax expression was associated with a transient slowing in cell-cycle progression and proliferation, increased apoptosis, and enhanced activation of the DNA damage response pathways. Longer-term follow-up (14 days) revealed an increase in the proportion of proliferating cells and a decrease in the fraction of apoptotic cells as the cells ceased Tax expression, resulting in a greater net expansion of the initially Tax-positive population. Time-lapse live-cell imaging showed enhanced cell-to-cell adhesion among Tax-expressing cells, and decreased cell motility of Tax-expressing cells at the single-cell level. The results demonstrate the within-clone and between-clone heterogeneity in the dynamics and patterns of HTLV-1 plus-strand transcriptional bursts and the balance of positive and negative consequences of the burst for the host cell.

The impact of HTLV-1 expression on the 3D structure and expression of host chromatin

“…To exclude the possibility that the anti-Tax antibody used in intracellular staining altered chromatin looping, we performed q4C assays using T-cell clones transduced with a reporter construct which expresses a modified EGFP with a half-life of ~2h (d2EGFP-TBX4B) when stimulated by Tax protein [ 11 ]. The GFP signal intensity was positively correlated with Tax expression [ 12 ]. Provirus-expressing (GFP + ) cells and non-expressing (GFP − ) cells were flow-sorted after fixation in 1% paraformaldehyde and subjected to the q4C assay.…”

“…While the transcriptional burst of tax is short-lived, Tax protein persists for some days in each cell. In the present experiment, the cells were selected on the basis of Tax protein expression: at any one time, therefore, the percentage of Tax protein-positive cells at any instant in these clones in vitro is relatively high [ 11 , 12 ].…”

“…By contrast, the minus strand is expressed in approximately 50% of circulating infected T cells at a given time [8]. Each HTLV-1-infected T-cell clone has its own pattern of proviral expression, that is, the frequency, intensity and duration of the transient transcriptional burst of the proviral plus-strand [10][11][12]. Since the HTLV-1 proviral sequence varies little within the host, these clone-specific differences are thought to be largely due to the unique genomic integration site of the provirus; other factors may include the antigen specificity and epigenetic modifications of the host cell.…”

The impact of HTLV-1 expression on the 3D structure and expression of host chromatin

“…To exclude the possibility that the anti-Tax antibody used in intracellular staining altered chromatin looping, we performed q4C assays using Tcell clones transduced with a reporter construct which expresses a modified EGFP with a half-life of ~2h (d2EGFP-TBX4B) when stimulated by Tax protein [11]. The GFP signal intensity was positively correlated with Tax expression [12]. Provirus-expressing (GFP + ) cells and non-expressing (GFP − ) cells were flow-sorted after fixation in 1% Next, we asked whether the aberrant host transcription is associated with proviral transcription or simply with the presence of the provirus, regardless of transcription.…”

Understanding the Immunopathology of HTLV-1-Associated Adult T-Cell Leukemia/Lymphoma: A Comprehensive Review

“…At the onset of ATL, approximately 50% of cases do not show Tax expression due to methylation or deletion of the 5′ LTR region; thus, selective proliferation of clones that are not attacked by CTLs is likely to be promoted [22]. In addition, recent studies have revealed that sense-side transcription leading to Tax expression occurs in transient bursts even in the absence of genomic deletions or epigenetic changes in the provirus [23][24][25]. This may be a viral survival strategy that minimizes Tax expression and avoids CTL attacks.…”