Epigenetic Silencing of Human Immunodeficiency Virus (HIV) Transcription by Formation of Restrictive Chromatin Structures at the Viral Long Terminal Repeat Drives the Progressive Entry of HIV into Latency
Epigenetic Silencing of Human Immunodeficiency Virus (HIV) Transcription by Formation of Restrictive Chromatin Structures at the Viral Long Terminal Repeat Drives the Progressive Entry of HIV into Latency
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DOI：
10.1128/jvi.01383-08
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发表时间：
2008-12-15
影响因子：
5.4
通讯作者：
Karn, Jonathan
中科院分区：
文献类型：
--
作者：
Pearson, Richard;Kim, Young Kyeung;Karn, Jonathan
人类免疫缺陷病毒（HIV）进入潜伏期的分子机制知之甚少。用顺式表达达特的慢病毒载体感染Jurkat T细胞后，基因表达逐渐沉默。当慢病毒载体携带具有H13 L突变的减毒达特基因时，沉默大大增强。慢病毒感染细胞的单个克隆显示出广泛的关闭率，其中大多数在30至80天之间显示出50%的沉默频率。沉默的克隆特征性地含有一小部分（0至15%）的继续表达d2 EGFP的活化细胞。当d2 EGFP（+）和d2 EGFP（-）细胞群从关闭的克隆中分离出来时，它们很快恢复到原来的非活性和活性细胞的分布，这表明d2 EGFP（+）细胞是由基因表达的随机波动引起的。使用染色质免疫沉淀（ChIP）测定对转录起始和延伸的详细分析证实，达特水平在潜伏感染的细胞中受到限制，但在前病毒再活化期间逐渐升高。使用潜伏感染细胞的克隆的ChIP测定证明潜伏前病毒携带高水平的脱乙酰化组蛋白和三甲基化组蛋白。相比之下，细胞基因I κ B α和GAPDH具有高水平的乙酰化组蛋白，而没有三甲基化组蛋白。与HIV前病毒相关的三甲基化组蛋白H3和HP 1-α的水平在肿瘤坏死因子α激活后迅速下降。感染后HIV转录的逐步关闭表明，靶向染色质结构的表观遗传机制选择性地限制HIV转录起始。这使达特的产量降低到维持HIV基因表达所需的水平以下。
The molecular mechanisms utilized by human immunodeficiency virus (HIV) to enter latency are poorly understood. Following the infection of Jurkat T cells with lentiviral vectors that express Tat in cis, gene expression is progressively silenced. Silencing is greatly enhanced when the lentiviral vectors carry an attenuated Tat gene with the H13L mutation. Individual clones of lentivirus-infected cells showed a wide range of shutdown rates, with the majority showing a 50% silencing frequency between 30 to 80 days. The silenced clones characteristically contained a small fraction (0 to 15%) of activated cells that continued to express d2EGFP. When d2EGFP(+) and d2EGFP(-) cell populations were isolated from the shutdown clones, they quickly reverted to the original distribution of inactive and active cells, suggesting that the d2EGFP(+) cells arise from stochastic fluctuations in gene expression. The detailed analysis of transcription initiation and elongation using chromatin immunoprecipitation (ChIP) assays confirms that Tat levels are restricted in the latently infected cells but gradually rise during proviral reactivation. ChIP assays using clones of latently infected cells demonstrate that the latent proviruses carry high levels of deacetylated histones and trimethylated histones. In contrast, the cellular genes I kappa B alpha and GAPDH had high levels of acetylated histones and no trimethylated histones. The levels of trimethylated histone H3 and HP1-alpha associated with HIV proviruses fell rapidly after tumor necrosis factor alpha activation. The progressive shutdown of HIV transcription following infection suggests that epigenetic mechanisms targeting chromatin structures selectively restrict HIV transcription initiation. This decreases Tat production below the levels that are required to sustain HIV gene expression.