Epigenetic regulation of HIV latency

描述（由申请人提供）：摘要潜伏性储库的存在阻碍了成功接受抗逆转录病毒治疗的感染患者中人类免疫缺陷病毒（HIV）的根除。我们最近报道了HIV启动子的DNA甲基化在HIV潜伏期的建立和维持中起着重要作用。我们还发现DNA甲基化抑制剂5-氮杂-2 '脱氧胞苷（aza-CdR）与NF-B激活剂协同作用，重新激活潜伏的HIV。我们建议进一步验证这一假设，即HIV启动子的甲基化在HIV潜伏期中起着关键作用，并且抑制DNA甲基化代表了一种新的治疗方法来诱导潜伏HIV的重新激活。由于可卡因已经成为一种可以改变表观遗传调控过程的分子，我们也计划研究它对HIV潜伏期的影响。我们的具体计划是：目标1。目的了解HIV启动子甲基化沉默的机制。我们将使用染色质免疫沉淀和shRNA介导的干扰来探索被招募到甲基化DNA的多蛋白复合物在HIV潜伏期中的作用：NuRD/MBD 2，SWI/SNF/Sin 3/MeCP 2和SUV 39 H1/HP 1/MBD 1复合物。目标2.确定与甲基化抑制剂协同作用重新激活潜伏HIV的小分子和细胞因子。我们将筛选与aza-CdR协同作用以重新激活潜伏的HIV的小分子和cDNA。在HIV潜伏期的J-Lat模型中与Aza-CdR协同作用以重新激活HIV的试剂将在潜伏期的原代细胞模型和来自HIV阳性供体的潜伏感染细胞中进行测试。目标3.目的研究HIV感染者淋巴细胞DNA甲基化。为了检验这一假设，即HIV整合在高甲基化的CpG岛或其附近导致潜伏期，我们建议比较静息和活化淋巴细胞中潜伏HIV整合位点的DNA甲基化状态，与J-Lat模型和原代人淋巴细胞中潜伏HIV的生产位点相比。目标4。研究HIV感染患者潜伏池的大小，甲基化抑制剂对其的再激活以及可卡因使用对这些变量的影响。我们建议使用体外HIV潜伏期的初级淋巴系统（与Vicente Planelles博士合作）和从HIV感染患者（可卡因使用者和非使用者）中分离的潜伏感染细胞中检测潜伏感染细胞中潜伏HIV的协同再激活。

DESCRIPTION (provided by applicant): ABSTRACT The presence of latent reservoirs has prevented the eradication of Human Immunodeficiency Virus (HIV) from infected patients successfully treated with antiretroviral therapy. We have recently reported that DNA methylation of the HIV promoter plays an important role in the establishment and maintenance of HIV latency. We also found that the inhibitor of DNA methylation, 5-aza-2'deoxycytidine (aza-CdR), synergizes with NF-B activators to reactivate latent HIV. We propose to further test the hypothesis that methylation of the HIV promoter plays a critical role in HIV latency and that inhibition of DNA methylation represents a novel therapeutic approach to induce the reactivation of latent HIV. Since cocaine has emerged as a molecule that can modify epigenetic regulatory processes we also plan to examine its effect in HIV latency. Our specific plans are to: Aim 1. To understand the mechanism of HIV promoter silencing via DNA methylation. We will use chromatin immunoprecipitation and shRNA-mediated interference to explore the roles in HIV latency of the multiprotein complexes that are recruited to methylated DNA: NuRD/MBD2, SWI/SNF/Sin3/MeCP2 and the SUV39H1/HP1/MBD1 complexes. Aim 2. To identify small molecules and cellular factors that reactivate latent HIV in synergy with methylation inhibitors. We will screen for small molecules and cDNAs that synergize with aza-CdR to reactivate latent HIV. Agents that synergize with Aza-CdR to reactivate HIV in the J-Lat model of HIV latency will be tested in a primary cell models of latency and in latently infected cells from HIV-positive donors. Aim 3. To study HIV DNA methylation in primary lymphocytes. To test the hypothesis that HIV integration in or near hypermethylated CpG islands leads to latency, we propose to compare the state of DNA methylation in resting and activated lymphocytes at the sites of latent HIV integration in comparison to productive sites both in the J-Lat model and in latent HIV in primary human lymphoid cells. Aim 4. To study the size of the latent pool in HIV-infected patients, its reactivation by methylation inhibitors and the effect of cocaine use on these variables. We propose to test for synergistic reactivation of latent HIV in latently infected cells using a primary lymphoid system for HIV latency in vitro (in collaboration with Dr, Vicente Planelles) and in latently-infected cells isolated from HIV-infected patients both cocaine users and non users.