Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Room W200, Cleveland, OH 44106-4960, USA.
J Mol Biol. 2011 Jul 29;410(5):896-916. doi: 10.1016/j.jmb.2011.03.054.
Latent human immunodeficiency virus (HIV) proviruses are thought to be primarily reactivated in vivo through stimulation of the T-cell receptor (TCR). Activation of the TCR induces multiple signal transduction pathways, leading to the ordered nuclear migration of the HIV transcription initiation factors NF-κB (nuclear factor κB) and NFAT (nuclear factor of activated T-cells), as well as potential effects on HIV transcriptional elongation. We have monitored the kinetics of proviral reactivation using chromatin immunoprecipitation assays to measure changes in the distribution of RNA polymerase II in the HIV provirus. Surprisingly, in contrast to TNF-α (tumor necrosis factor α) activation, where early transcription elongation is highly restricted due to rate-limiting concentrations of Tat, efficient and sustained HIV elongation and positive transcription elongation factor b (P-TEFb) recruitment are detected immediately after the activation of latent proviruses through the TCR. Inhibition of NFAT activation by cyclosporine had no effect on either HIV transcription initiation or elongation. However, examination of P-TEFb complexes by gel-filtration chromatography showed that TCR signaling led to the rapid dissociation of the large inactive P-TEFb:7SK RNP (small nuclear RNA 7SK ribonucleoprotein) complex and the release of active low-molecular-weight P-TEFb complexes. Both P-TEFb recruitment to the HIV long terminal repeat and enhanced HIV processivity were blocked by the ERK (extracellular-signal-regulated kinase) inhibitor U0126, but not by AKT (serine/threonine protein kinase Akt) and PI3K (phosphatidylinositol 3-kinase) inhibitors. In contrast to treatment with HMBA (hexamethylene bisacetamide) and DRB (5,6-dichlorobenzimidazole 1-β-ribofuranoside), which disrupt the large 7SK RNP complex but do not stimulate early HIV elongation, TCR signaling provides the first example of a physiological pathway that can shift the balance between the inactive P-TEFb pool and the active P-TEFb pool and thereby stimulate proviral reactivation.
潜伏的人类免疫缺陷病毒 (HIV) 前病毒被认为主要通过 T 细胞受体 (TCR) 的刺激在体内被重新激活。TCR 的激活诱导多种信号转导途径，导致 HIV 转录起始因子 NF-κB（核因子 κB）和 NFAT（激活 T 细胞的核因子）的有序核迁移，以及对 HIV 转录延伸的潜在影响。我们使用染色质免疫沉淀测定法监测前病毒重新激活的动力学，以测量 HIV 前病毒中 RNA 聚合酶 II 的分布变化。令人惊讶的是，与 TNF-α（肿瘤坏死因子-α）激活相反，由于 Tat 的限速浓度，早期转录延伸受到高度限制，通过 TCR 激活潜伏前病毒后，立即检测到有效的和持续的 HIV 延伸和正转录延伸因子 b（P-TEFb）募集。环孢素抑制 NFAT 激活对 HIV 转录起始或延伸均无影响。然而，通过凝胶过滤色谱法对 P-TEFb 复合物进行检查表明，TCR 信号导致大的无活性 P-TEFb：7SK RNP（小核 RNA 7SK 核糖核蛋白）复合物的快速解离，并释放出活性低分子量 P-TEFb 复合物。P-TEFb 募集到 HIV 长末端重复序列和增强 HIV 进程都被 ERK（细胞外信号调节激酶）抑制剂 U0126 阻断，但不是通过 AKT（丝氨酸/苏氨酸蛋白激酶 Akt）和 PI3K（磷酸肌醇 3-激酶）抑制剂。与 HMBA（己二亚甲基双乙酰）和 DRB（5,6-二氯苯并咪唑 1-β-核糖呋喃苷）的治疗不同，后者破坏大 7SK RNP 复合物但不刺激早期 HIV 延伸，TCR 信号提供了第一个生理途径的例子，可以改变无活性 P-TEFb 池和活性 P-TEFb 池之间的平衡，从而刺激前病毒重新激活。
