Epigenetic regulation of HIV latency

描述(由申请人提供)摘要：潜伏蓄水池的存在阻碍了成功接受抗逆转录病毒治疗的感染患者根除人类免疫缺陷病毒(HIV)。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的作用。我们还发现DNA甲基化的抑制剂5-氮杂-2‘脱氧胞苷(aza-CDR)与核因子-B激活剂协同作用重新激活潜伏的HIV。我们建议进一步检验这一假设，即HIV启动子的甲基化在HIV潜伏时间中起关键作用，并且抑制DNA甲基化代表着诱导潜伏HIV重新激活的一种新的治疗方法。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，我们还计划检查它在艾滋病毒潜伏期中的作用。我们的具体计划是：目的1.了解DNA甲基化导致HIV启动子沉默的机制。我们将使用染色质免疫沉淀和shRNA介导的干扰来探索招募到甲基化DNA的多蛋白复合体：NuRD/Mbd2、SWI/SNF/Sin3/MeCP2和SUV39H1/HP1/MBD1复合体在HIV潜伏期中的作用。目的2.鉴定与甲基化抑制剂协同作用使潜伏的HIV重新激活的小分子和细胞因子。我们将筛选与aza-CDR协同作用的小分子和cDNA，以重新激活潜伏的HIV。在潜伏期的J-Lat模型中，与aza-CDR协同激活HIV的药物将在潜伏期的初级细胞模型和HIV阳性捐赠者的潜伏感染细胞中进行测试。目的3.研究人类免疫缺陷病毒(HIV)初级淋巴细胞DNA甲基化情况。为了验证HIV整合在超甲基化的CpG岛上或附近导致潜伏期的假设，我们建议比较J-Lat模型和原代人类淋巴细胞中潜伏的HIV的产生部位的静止和激活的淋巴细胞中的DNA甲基化状态。目的4.研究HIV感染患者潜伏池的大小、甲基化抑制剂对潜伏池的重新激活以及可卡因的使用对这些变量的影响。我们建议使用HIV潜伏期的初级淋巴系统(与Vicente Planelle博士合作)在体外测试潜伏感染细胞中潜伏艾滋病毒的协同重新激活，并在从吸食可卡因和不吸食可卡因的HIV感染患者分离的潜伏感染细胞中进行测试。公共卫生相关性：叙述对潜伏的艾滋病毒基因组的DNA甲基化和其他表观遗传修饰的鉴定突出了抑制艾滋病毒转录的新机制。DNA甲基化抑制剂aza-DCR与前列环素或肿瘤坏死因子-1协同激活潜伏的HIV的证据表明，表观遗传修饰可以作为我们重新激活潜伏的HIV的药物靶点。最近的实验还表明，使用可卡因可以在大脑中诱导长期的表观遗传修饰，可能还会在免疫系统中诱导。在这里，我们建议研究表观遗传修饰在HIV潜伏期中的作用，以及改变表观遗传沉默的药物作为一种新的治疗方法来诱导潜伏的HIV重新激活的可能作用。

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.