Epigenetic regulation of HIV latency

描述（由申请人提供）：潜伏病毒库的存在阻碍了人类免疫缺陷病毒（HIV）从成功接受抗逆转录病毒治疗的感染患者中根除。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-aza-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HIV启动子的甲基化在HIV潜伏期中起关键作用的假设，以及抑制DNA甲基化代表了一种诱导潜伏HIV再激活的新治疗方法。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。了解HIV启动子通过DNA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：NuRD/MBD2， SWI/SNF/Sin3/MeCP2和SUV39H1/HP1/MBD1复合物在HIV潜伏期中的作用。目标2。鉴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HIV的小分子和cdna。在J-Lat HIV潜伏期模型中，与Aza-CdR协同重新激活HIV的药物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：研究原发性淋巴细胞中HIV DNA甲基化。为了验证HIV整合在高甲基化CpG岛或其附近导致潜伏期的假设，我们建议比较J-Lat模型和初代人淋巴细胞中潜伏HIV整合位点的DNA甲基化状态和激活淋巴细胞中潜伏HIV整合位点与产生位点的DNA甲基化状态。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。我们建议在体外（与Vicente Planelles博士合作）和从可卡因使用者和非使用者HIV感染患者中分离的潜伏感染细胞中，使用用于HIV潜伏期的原发性淋巴系统测试潜伏HIV在潜伏感染细胞中的协同再激活。DNA甲基化和潜伏HIV基因组的其他表观遗传修饰的鉴定突出了抑制HIV转录的新机制。DNA甲基化抑制剂aza-DcR与prostratin或TNF-协同作用重新激活潜伏性HIV的研究表明，表观遗传修饰可以作为我们重新激活潜伏性HIV的药物靶点。最近的实验还表明，可卡因的使用可以在大脑和可能在免疫系统中诱导长期的表观遗传修饰。在这里，我们建议研究表观遗传修饰在HIV潜伏期中的作用，以及修饰表观遗传沉默的药物作为诱导潜伏HIV再激活的新治疗方法的可能作用。

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. PUBLIC HEALTH RELEVANCE: NARRATIVE The identification of DNA methylation and other epigenetic modifications of the latent HIV genome highlights a novel mechanism for the suppression of HIV transcription. The demonstration that the DNA methylation inhibitor, aza-DcR, synergizes with prostratin or TNF- to reactivate latent HIV suggest that epigenetic modification could be used as a drug target in our effort to reactivate latent HIV. Recent experiments also indicate that cocaine use can induce prolonged epigenetic modifications in the brain and possibly in the immune system. Here we propose to study the role of epigenetic modifications in HIV latency and the possible role of drugs that modify epigenetic silencing as a novel therapeutic approach to induce the reactivation of latent HIV.