Latent HIV-1 reactivation in transgenic mice requires cell cycle-dependent demethylation of CREB/ATF sites in the LTR
Latent HIV-1 reactivation in transgenic mice requires cell cycle-dependent demethylation of CREB/ATF sites in the LTR
复制标题
DOI：
10.1097/00002030-200301240-00005
复制
发表时间：
2003-01-24
期刊：
影响因子：
3.8
通讯作者：
Iwakura, Y
中科院分区：
文献类型：
--
作者：
Tanaka, J;Ishida, T;Iwakura, Y
目的：我们以前生产了一系列的转基因小鼠携带HIV-1基因组中缺乏pol基因。虽然在正常生理条件下淋巴细胞中的HIV-1基因组处于休眠状态，但它可以通过诱导白细胞介素-1 α/β和肿瘤坏死因子-α在体内被脂多糖（LPS）重新激活。在这份报告中，我们进一步分析了使用这种转基因小鼠model.Design和方法的潜伏HIV-1的再激活机制：可能参与的CpG甲基化在HIV-1潜伏期进行了检查，通过处理转基因淋巴细胞与去甲基化剂，5 '-氮杂胞苷。使用亚硫酸氢盐基因组测序方法分析HIV-1长末端重复序列（LTR）中的CpG甲基化。由于以前的研究表明，CpG去甲基化依赖于细胞周期的进展，我们分析了细胞周期的进展和LPS诱导的HIV-1的再激活标记淋巴细胞与细胞内的荧光素，羧基荧光素二乙酸琥珀酰亚胺酯之间的关系：我们发现，5 '-氮杂胞苷增强HIV-1的表达9倍相比，单独用LPS治疗。此外，仅在经历细胞分裂的细胞中观察到LPS对HIV-1 p24的诱导，而在细胞周期进程被含羞草碱、阿非迪霉素或诺考达唑阻断的细胞中，诱导被阻止。LPS诱导的HIV-1再激活与位于CREB/ATF结合位点的两个CpG位点的去甲基化在HIV-1 LTR中以细胞周期依赖的方式进行相关。结论：这些观察表明，细胞周期进程依赖的CREB/ATF位点的去甲基化在LTR中是潜伏的HIV-1基因组在转基因小鼠中的再激活的关键。(C)2003年利平科特威廉姆斯威尔金斯。
Objective: We previously produced a line of transgenic mice that carried the HIV-1 genome deficient in the pol gene. Although the HIV-1 genome in the lymphocytes was dormant under normal physiological conditions, it could be reactivated in vivo by lipopolysaccharide (LPS) administration via induction of interleukin-1alpha/beta and tumour necrosis factor-alpha. In this report, we analysed further the reactivation mechanism of the latent HIV-1 using this transgenic mouse model.Design and methods: Possible involvement of CpG methylation in HIV-1 latency was examined by treating transgenic lymphocytes with a demethylating agent, 5'-azacytidine. CpG methylation in the HIV-1 long terminal repeat (LTR) was analysed using the bisulfite genomic sequencing method. As previous studies suggested that CpG demethylation depended on the cell cycle progression, we analysed the relation between cell cycle progression and LPS-induced reactivation of HIV-1 by labelling lymphocytes with an intracellular fluorescein, carboxyfluorescein diacetate succinimidyl ester.Results: We found that 5'-azacytidine enhanced HIV-1 expression ninefold compared to treatment with LPS alone. Furthermore, HIV-1 p24 induction by LPS was observed only in cells that had undergone cell division, while induction was prevented in cells in which cell cycle progression was blocked either by mimosine, aphidicolin, or nocodazole. LPS-induced HIV-1 reactivation was associated with demethylation of two CpG sites located in the CREB/ATF binding sites in the HIV-1 LTR in a cell cycle-dependent manner.Conclusions: These observations indicate that cell cycle progression-dependent demethylation of the CREB/ATF sites in the LTR is crucial for the reactivation of latent HIV-1 genome in transgenic mice. (C) 2003 Lippincott Williams Wilkins.