A histone deacetylase network regulates epigenetic reprogramming and viral silencing in HIV infected cells

During HIV infection, a latent viral reservoir is formed that persists during antiretroviral therapy (ART) and is maintained by a heritable state of transcriptional repression. Recent findings indicate that much of the reservoir originates from infections occurring in the weeks near the time of ART initiation, raising the important possibility that interventions during this period might prevent reservoir seeding and substantially reduce reservoir size. Based on this concept, we tested the ability of compounds that target epigenetic machinery to prevent the establishment of latent HIV infection in primary CD4 T cells. We identified class 1 histone deacetylase inhibitors (HDACi) as potent agents of latency prevention, an activity distinct from latency reversal. Inhibiting HDACs in productively infected cells caused extended maintenance of HIV expression, even after HDACi withdrawal, and this activity was associated with persistently elevated H3K9 acetylation and reduced H3K9 methylation at the viral LTR promoter region. HDAC inhibition in HIV-infected CD4 T cells during effector-to-memory transition led to a striking change in the memory subset distribution indicating reprogramming of cell identity. Through knockout of individual HDACs and use of HDAC-selective inhibitors, we determined that HDAC1 and HDAC3 play crucial and distinct roles in proviral silencing initiation. Overall, this work indicates that a network of HDACs regulate a critical gateway process for HIV latency establishment and are required for the development of CD4 T-cell memory subsets that preferentially harbor long-lived, latent provirus. Epigenetic reprogramming by clinical targeting of HDACs during ART initiation may represent a novel way to prevent seeding of the HIV reservoir in vivo CUT&RUN profiling of H3K4me3, H3K27ac, H3K9ac, H3K9me3 and H3K27me3 histone marks in infected cells that had been cultured with or without vorinostat (SAHA). We similarly profiled total RNA Polymerase II (RPD1 subunit, tPol2) and RNA Polymerase II phosphorylated on Ser 2 of the heptameric repeat region (P-S2, Pol2s2)

在人类免疫缺陷病毒（HIV）感染过程中，会形成潜伏病毒库，该病毒库在抗逆转录病毒治疗（ART）期间持续存在，并由可遗传的转录抑制状态维持。近期研究显示，该潜伏病毒库的大部分源自抗逆转录病毒治疗启动前后数周内发生的感染，这带来了一项重要潜在可能：在此阶段实施干预或可阻断病毒库定植，并大幅缩减病毒库规模。基于这一理念，我们测试了靶向表观遗传机制的化合物在原代CD4 T细胞中预防潜伏HIV感染建立的能力。我们鉴定出I类组蛋白去乙酰化酶抑制剂（HDACi）可作为强效的潜伏感染预防制剂，其活性与潜伏逆转作用截然不同。在产毒感染的细胞中抑制组蛋白去乙酰化酶，可使HIV的表达持续维持，即便在撤除HDACi后亦是如此；该活性与病毒长末端重复序列（LTR）启动子区域的H3K9乙酰化水平持续升高、H3K9甲基化水平降低密切相关。在效应T细胞向记忆T细胞转化期间对HIV感染的CD4 T细胞进行组蛋白去乙酰化酶抑制，会使记忆细胞亚群分布发生显著改变，表明细胞身份已被重编程。通过单一组蛋白去乙酰化酶敲除实验以及选择性HDAC抑制剂的应用，我们证实HDAC1与HDAC3在前病毒沉默启动过程中发挥着关键且独特的作用。综上，本研究表明，组蛋白去乙酰化酶网络调控着HIV潜伏建立的关键门户过程，且对于优先携带长寿潜伏前病毒的CD4 T细胞记忆亚群的发育不可或缺。在抗逆转录病毒治疗启动阶段，通过临床靶向组蛋白去乙酰化酶实施表观遗传重编程，或可成为一种在体内阻断HIV病毒库定植的全新策略。我们对分别在添加或不添加伏立诺他（Vorinostat, SAHA）的培养基中培养的感染细胞，开展了H3K4me3、H3K27ac、H3K9ac、H3K9me3及H3K27me3组蛋白修饰标记的CUT&RUN图谱分析。我们同样对总RNA聚合酶II（RPD1亚基，tPol2）以及七聚体重复区域第2位丝氨酸磷酸化的RNA聚合酶II（P-S2, Pol2s2）进行了图谱分析。