Thermal cycler protocol for qPCR.

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 essential for 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 the virus, 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.

人类T细胞白血病病毒1型（HTLV-1）通过整合至CD4阳性T细胞（CD4+ T细胞）的基因组中，可在宿主终身持续存在。前病毒基因表达对于前病毒的存活及前病毒载量的维持至关重要，其可通过诱导受感染细胞发生促增殖变化实现上述功能。尽管这类促增殖变化在HTLV-1感染过程中发挥关键作用，且宿主体内会持续产生针对该病毒的细胞毒性T淋巴细胞应答，但从外周血分离的新鲜细胞中，极少能检测到有义链来源的前病毒转录本；近期研究表明，在特定时间点仅少量细胞亚群会间歇性表达此类转录本。对受感染细胞进行体外培养，可促使外周血来源的受感染细胞同步激活前病毒表达，从而为研究原代细胞中的病毒再激活相关因子提供了可行途径。本研究采用批量RNA测序（bulk RNA-seq）技术，对源自非恶性HTLV-1感染受试者的原代外周血CD4阳性T细胞，在体外完成前病毒再激活后的6天内的宿主转录组进行了检测分析。受感染细胞展现出保守的再激活应答，其特征为依次呈现基因表达、细胞分裂以及后续的病毒水平传播等多个离散阶段。我们观察到，病毒再激活后宿主细胞的多梳基因（Polycomb gene）表达发生广泛改变，包括多梳抑制复合体2（PRC2）的转录本水平升高，以及多梳抑制复合体1（PRC1）各组分的表达出现多样变化。我们推测，此类转录变化构成了负反馈环路：在前病毒表达结束后，通过重新沉积H2AK119ub1修饰，从而维持前病毒的潜伏状态。通过RNA干扰（RNAi）实验，我们发现部分去泛素化酶——包括BAP1、USP14和OTUD5——均可促进前病毒的转录。本研究数据清晰阐明了原代HTLV-1携带者淋巴细胞中HTLV-1前病毒再激活的详细动态轨迹，以及该过程对宿主细胞产生的影响。