Biology subjects

Ramanayake, S.

Publications and source records attributed to Ramanayake, S..

2 recordsLinked to original sources

bioRxiv2022

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

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 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 decreased proliferation, increased apoptosis, enhanced activation of the DNA damage response pathways, and slower cell-cycle progression. 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. Author SummaryHuman T-cell leukaemia virus type 1 (HTLV-1) causes disabling or fatal diseases in up to 10% of the infected individuals. The expression of viral protein Tax is essential to cause new infections and contributes to HTLV-1-associated diseases. The proviral plus-strand, which encodes Tax, is expressed in intense intermittent bursts. However, the kinetics of Tax expression and its short and longer-term impact on the infected cell are not well understood. We combined live-cell imaging and mathematical modelling to study Tax expression kinetics in two naturally-infected T-cell clones. Single-cell analysis showed five patterns of Tax expression, with most Tax-positive cells expressing continuously during the 30-hour imaging. The average duration of Tax expression in the two clones was 94 and 417 hours respectively, by mathematical modelling. Tax expression correlated with decreased proliferation, increased apoptosis, enhanced activation of DNA damage response pathways and delayed progression through the cell-cycle. Extended observation showed an increase in the proportion of proliferating cells and a decrease in the percentage of apoptotic cells as cells ceased Tax expression, resulting in a greater net growth of the originally Tax-positive population. Tax-expressing cells also formed cell clumps and showed reduced cell movement. This study highlights prolonged Tax expression and its short and longer-term impact on naturally-infected cells.

microbiology↗

bioRxiv2022

Time-course of host cell transcription during the HTLV-1 transcriptional burst

The human T-cell leukemia virus type 1 (HTLV-1) transactivator protein Tax has pleiotropic functions in the host cell affecting cell-cycle regulation, DNA damage response pathways and apoptosis. These actions of Tax have been implicated in the persistence and pathogenesis of HTLV-1-infected cells. It is now known that tax expression occurs in transcriptional bursts of the proviral plus-strand, but the effects of the burst on host transcription are not fully understood. We carried out RNA sequencing of two naturally-infected T-cell clones transduced with a Tax-responsive Timer protein, which undergoes a time-dependent shift in fluorescence emission, to study transcriptional changes during successive phases of the HTLV-1 plus-strand burst. We found that the transcriptional regulation of genes involved in the NF-{kappa}B pathway, cell-cycle regulation, DNA damage response and apoptosis inhibition were immediate effects accompanying the plus-strand burst, and are limited to the duration of the burst. The results distinguish between the immediate and delayed effects of HTLV-1 reactivation on host transcription, and between clone-specific effects and those observed in both clones. The major transcriptional changes in the infected host T-cells observed here, including NF-kB, are transient, suggesting that these pathways are not persistently activated at high levels in HTLV-1-infected cells. The two clones diverged strongly in their expression of genes regulating the cell cycle. Up-regulation of senescence markers was a delayed effect of the proviral plus-strand burst and the up-regulation of some pro-apoptotic genes outlasted the burst. We found that activation of the arylhydrocarbon receptor (AhR) pathway enhanced and prolonged the proviral burst, but did not increase the rate of reactivation. Our results also suggest that sustained plus-strand expression is detrimental to the survival of infected cells. Author SummaryHuman T-cell leukemia virus type 1 (HTLV-1) causes a lifelong infection that results in disease in [~]10% of cases. The HTLV-1 transactivator protein Tax is involved in both the persistence of infected host cells, and the pathogenesis of HTLV-1 infection. tax is transcribed from the plus-strand of the provirus, and tax expression is not constitutive, but limited to transcriptional bursts. How these bursts affect host cell transcription is not completely understood. Here, we studied the temporal changes in host transcription during successive phases of the plus-strand burst in two naturally-infected T-cell clones. We found that the deregulation of genes involved in Tax-associated processes, including NF-{kappa}B activation, cell-cycle regulation, DNA damage response and suppression of apoptosis, coincided with the early phase of the plus-strand burst: these transcriptional effects appear to be limited to the duration of the proviral plus-strand expression. Regulation of cell-cycle genes diverged between the clones, demonstrating the heterogeneity of naturally-infected cells. We observed a pro-apoptotic response, which outlasted the burst and may indicate increased risk of apoptosis following the burst. Finally, we observed that AhR activity regulated the intensity and duration of the burst, but not the dynamics of reactivation.

microbiology↗