Investigating RNA polymerase III driven mechanisms in regulating HIV latency

摘要 静息的CD4+T细胞携带大部分潜伏的人类免疫缺陷病毒(HIV) 感染。消除这种水库是一项极具挑战性的任务，因为涉及 调节HIV潜伏期的多种机制。因此，也许目前没有一个 有希望的潜伏期反转剂(LRA)能够减少潜伏期前病毒的大小 病人体内的蓄水池。因此，迫切需要寻找新的机制和治疗靶点。 可以有针对性地清除非均质潜伏油藏。RNA聚合酶III(RNA Pol III) 似乎是一个主控调节器，具有通过中介调节潜伏期的未知潜力 不同的机制，如i)调节邻近RNA POL II基因的表达 启动子和ii)转录新的非编码RNA，可能调节其表达 细胞/病毒基因。我们的初步研究表明，Pol III转录的富集物 潜伏细胞中的非编码RNA，即散布在AlU之间的7SK、21A和BC200 重复着。这与HIV潜伏期高度相关，因为HIV基因组被发现优先于 Alu附近的整合重复。因此，使用RNA POLIII抑制剂ML60218导致 潜伏细胞系J89GFP和THP89GFP史无前例的重新激活(高达90%)，在一个剂量- 依赖方式(25μM-50μM)。此外，我们还观察到高度的细胞死亡。 在艾滋病毒感染的细胞中，由于病毒的细胞病变效应，而未感染的细胞维持存活 即使在非常高浓度的RNA PolIII抑制剂(100μM)下也是如此。这些令人兴奋的发现和 将利用确凿的报告来检验RNA Pol III在 在建立HIV潜伏期和靶向新的RNA中间效应物中的作用 POL III驱动的机制可能会增强治愈策略的有效性。这项研究分为两个部分 分成两个具体目标，重点研究由基因组直接驱动的RNA Pol III 可调节艾滋病毒潜伏期的间接(通过ncRNA)机制。在目标1中，效果 RNA Pol III的抑制/敲除将在体外培养的原代CD4+T细胞模型中进行测试。 此外，在Aim 1.2中，我们将调查在附近存在的RNA Pol III是否会改变 HIV 5‘LTR染色质景观。在目标2中，我们将识别RNA Pol III转录的非编码 RNA-Seq与RNA Pol III CHIP-Seq相结合参与潜伏的RNA。 最后，将对选定的非编码RNA单独或在 研究它们在促进HIV 5‘LTR抑制染色质状态中的作用。我们的 这项研究具有很高的创新性，因为我们的目标是识别新的表观遗传调节剂，这些调节剂可以 同步有的放矢地克服与艾滋病毒休克和杀死策略相关的挑战 解药。这项研究的成功完成将提供关键的机械信息，这些信息可能 解决潜在储集层之间的非均质性。

ABSTRACT Resting CD4+ T cells harbor majority of latent Human Immunodeficiency Virus (HIV) during infection. Elimination of this reservoir is an extremely challenging task because of involvement of multiple mechanisms in regulating HIV latency. Therefore, perhaps, none of the currently promising latency reversing agents (LRAs) were able to reduce the size of the latent proviral reservoir in patients. Hence, there is a dire need to find novel mechanisms and therapeutic targets that can be targeted to purge the heterogenous latent reservoir. RNA Polymerase III (RNA Pol III) appears to be a master regulator with unexplored potential of regulating latency via mediating distinct mechanisms, such as i) regulating the expression from neighboring RNA Pol II gene promoters and ii) transcription of novel noncoding RNAs with potential to regulate expression of cellular/viral genes. Our preliminary studies suggest the enrichment of Pol III transcribed noncoding RNAs in latent cells, namely 7SK, 21A and BC200 that are interspersed among Alu repeats. This is highly relevant to HIV latency because the HIV genome is found to preferentially integrate near Alu repeats. Consequently, use of an RNA Pol III inhibitor, ML60218, resulted in an unprecedented reactivation (up to 90%) of latent cell lines J89GFP and THP89GFP, in a dose- dependent manner (25 μM-50 μM). Further, we observed a high degree of cell death specifically in HIV infected cells due to viral cytopathic effects, whereas uninfected cells maintained survival even at a very high concentration of RNA Pol III inhibitor (100 μM). These exciting findings and corroborating reports will be leveraged to test the hypothesis that RNA Pol III plays a crucial role in the establishment of HIV latency and targeting novel intermediate effectors of RNA Pol III driven mechanisms may enhance the efficacy of cure strategies. This study is divided into two specific aims that will be focused on investigating RNA Pol III driven direct (by genomic occupancy) and indirect (by ncRNAs) mechanisms that may regulate HIV latency. In Aim 1, effect of RNA Pol III inhibition/knockdown will be tested in ex vivo cultured primary CD4+ T cell model. Further in Aim 1.2 we will investigate if physical presence of RNA Pol III in proximity can modify chromatin landscape at HIV 5´ LTR. In Aim 2, we will identify RNA Pol III transcribed noncoding RNAs involved in latency by employing RNA-seq in combination with RNA Pol III ChIP-Seq. Finally, gene knockdown studies will be performed for select noncoding RNAs alone or in combination to investigate their role in promoting repressed chromatin state at HIV 5´ LTR. Our study is highly innovative as we aim to identify novel epigenetic modulators that can be synchronously targeted to overcome challenges associated with shock and kill strategy of HIV cure. Successful completion of this study will provide critical mechanistic information which may address the heterogeneity among latent reservoirs.