Biology subjects

Bangham, C. R. M.

Publications and source records attributed to Bangham, C. R. M..

6 recordsLinked to original sources

bioRxiv2023

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

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 aberrant 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.

microbiology↗

bioRxiv2023

The transcriptome of HTLV-1-infected primary cells following reactivation reveals changes to host gene expression central to the proviral life cycle

Infections by Human T cell Leukaemia Virus type 1 (HTLV-1) persist for the lifetime of the host by integrating into the genome of CD4+ T cells. Proviral gene expression is core to proviral survival and the maintenance of the proviral load, through the pro-proliferative changes it induces in infected cells. Despite their role in HTLV-1 infection and a persistent cytotoxic T lymphocyte response raised against them, proviral transcripts from the sense-strand are rarely detected in fresh cells extracted from the peripheral blood, and have recently been found to be expressed intermittently by a small subset of cells at a given time. Ex vivo culture of infected cells prompts synchronised proviral expression in infected cells from peripheral blood, allowing the study of factors involved in reactivation in primary cells. Here, we used bulk RNA-seq to examine the host transcriptome over six days in vitro, following proviral reactivation in primary peripheral CD4+ T cells isolated from subjects with non-malignant HTLV-1 infection. Infected cells displayed a conserved response to reactivation, characterised by discrete stages of gene expression, cell division and subsequently horizontal transmission of the virus. We observed widespread changes in Polycomb gene expression following reactivation, including an increase in PRC2 transcript levels and diverse changes in the expression of PRC1 components. We hypothesize that these transcriptional changes constitute a negative feedback loop that maintains proviral latency by re-deposition of H2AK119ub1 following the end of proviral expression. Using RNAi, we found that certain deubiquitinases, BAP1, USP14 and OTUD5 each promote proviral transcription. These data demonstrate the detailed trajectory of HTLV-1 proviral reactivation in primary HTLV-1-carrier lymphocytes and the impact on the host cell. Author summaryHuman T cell Leukemia Virus type I (HTLV-1) is a retrovirus which causes an aggressive leukemia or lymphoma, or a chronic inflammatory disease of the central nervous system, in a subset ({approx} 10%) of affected carriers. Whilst the virus is only intermittently expressed by infected cells, the virus persists in its host by increasing the proliferation rate and survival of infected cells. It is therefore imperative to understand the mechanisms that control the activation and deactivation of the virus. We examined the expression of host and viral genes during HTLV-1 reactivation in cells freshly isolated from patients blood. The infected cells displayed consistent changes in gene expression over six days. We observed changes in the expression of the Polycomb group of epigenetic modifiers, known to impact HTLV-1 transcription, which may form a negative-feedback mechanism allowing the virus to return to a latent (quiescent) state following activation. We additionally identified a set of three deubiquitinases that increase expression of the virus. Our data detail the changes in gene expression underlying core aspects of the HTLV-1 life cycle in primary cells, and provide a resource for further investigation.

microbiology↗

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↗

bioRxiv2021

Selective clonal persistence of human retroviruses in vivo: radial chromatin organization, integration site and host transcription

The human retroviruses HTLV-1 and HIV-1 persist in vivo, despite the host immune response and antiretroviral therapy, as a reservoir of latently infected T-cell clones. It is poorly understood what determines which clones survive in the reservoir and which are lost. We compared >160,000 HTLV-1 integration sites from T-cells isolated ex vivo from naturally-infected subjects with >230,000 integration sites from in vitro infection, to identify the genomic features that determine selective clonal survival. Three factors explained >40% of the observed variance in clone survival of HTLV-1 in vivo: the radial intranuclear position of the provirus, its absolute genomic distance from the centromere, and the intensity of host genome transcription flanking the provirus. The radial intranuclear position of the provirus and its distance from the centromere also explained ~7% of clonal persistence of HIV-1 in vivo. Selection for transcriptionally repressive nuclear compartments favours clonal persistence of human retroviruses in vivo.

microbiology↗

bioRxiv2018

The human leukemia virus HTLV-1 alters the structure and transcription of host chromatin in cis

Chromatin looping controls gene expression by regulating promoter-enhancer contacts, the spread of epigenetic modifications, and the segregation of the genome into transcriptionally active and inactive compartments. We studied the impact on the structure and expression of host chromatin by the human retrovirus HTLV-1. We show that HTLV-1 disrupts host chromatin structure by forming loops between the provirus and the host genome; certain loops depend on the critical chromatin architectural protein CTCF, which we recently showed binds to the HTLV-1 provirus. Finally, we show that the provirus causes two distinct patterns of abnormal transcription of the host genome in cis: bidirectional transcription in the host genome immediately flanking the provirus, and clone-specific transcription in cis at non-contiguous loci up to >300 kb from the integration site. We conclude that HTLV-1 causes insertional mutagenesis up to the megabase range in the host genome in >104 persistently-maintained HTLV-1+ T-cell clones in vivo.

microbiology↗