Investigating RNA polymerase III driven mechanisms in regulating HIV latency

研究 RNA 聚合酶 III 驱动机制调节 HIV 潜伏期

基本信息

  • 批准号:
    10484480
  • 负责人:
  • 金额:
    $ 49.53万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2022
  • 资助国家:
    美国
  • 起止时间:
    2022-02-01 至 2025-01-31
  • 项目状态:
    未结题

项目摘要

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.
摘要 静息的CD 4 + T细胞携带大多数潜伏的人类免疫缺陷病毒(HIV), 感染消除这一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 有前途的潜伏期逆转剂(LRA)能够减少潜伏前病毒的大小, 患者体内的储药器。因此,迫切需要寻找新的机制和治疗靶点 其可以被定向以清除非均质潜在储层。RNA聚合酶III(RNA Pol III) 似乎是一个主调节器,具有通过介导调节潜伏期的未开发潜力。 不同的机制,例如i)调节邻近RNA Pol II基因的表达 启动子和ii)新的非编码RNA的转录,所述新的非编码RNA具有调节 细胞/病毒基因。我们的初步研究表明,Pol III转录的富集 潜伏细胞中的非编码RNA,即散布在Alu中的7SK,21 A和BC 200 重复。这与HIV潜伏期高度相关,因为发现HIV基因组优先 整合近Alu重复。因此,使用RNA Pol III抑制剂ML 60218, 一个前所未有的重新激活(高达90%)的潜伏细胞系J89 GFP和THP 89 GFP,在剂量- 依赖性方式(25 μM-50 μM)。此外,我们观察到高度的细胞死亡, 在HIV感染的细胞中,由于病毒的细胞病变效应,而未感染的细胞保持存活 即使在非常高浓度的RNA Pol III抑制剂(100 μM)下也是如此。这些令人兴奋的发现和 证实报告将被用来检验假设,即RNA Pol III起着至关重要的作用。 在建立HIV潜伏期和靶向新型RNA中间效应物中的作用 Pol III驱动的机制可以增强治愈策略的功效。本研究分为 分为两个具体的目标,将集中在研究RNA Pol III驱动的直接(由基因组 占据)和间接(通过ncRNA)机制,可以调节HIV潜伏期。在目标1中, 将在离体培养的原代CD 4 + T细胞模型中测试RNA Pol III抑制/敲低。 进一步在目标1.2中,我们将研究邻近的RNA Pol III的物理存在是否可以修饰 HIV 5 ′ LTR的染色质景观。在目标2中,我们将鉴定RNA Pol III转录的非编码区, 通过采用RNA-seq与RNA Pol III ChIP-Seq组合,参与潜伏期的RNA。 最后,将对选择的非编码RNA单独或联合应用进行基因敲除研究。 联合研究它们在促进HIV 5 ′ LTR抑制染色质状态中的作用。我们 这项研究是高度创新的,因为我们的目标是确定新的表观遗传调节剂, 同步有针对性地克服与艾滋病毒的休克和杀死策略相关的挑战 疗方成功完成本研究将提供关键的机制信息, 解决潜在储层的非均质性问题。

项目成果

期刊论文数量(3)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Human immunodeficiency virus-1 Tat exerts its neurotoxic effects by downregulating Sonic hedgehog signaling.
  • DOI:
    10.1007/s13365-022-01061-8
  • 发表时间:
    2022-04
  • 期刊:
  • 影响因子:
    3.2
  • 作者:
    Khan, Irfan A.;Worrad, Arthur H.;Singh, Meera, V;Maggirwar, Sanjay B.;Singh, Vir B.
  • 通讯作者:
    Singh, Vir B.
Initiation of combined antiretroviral therapy confers suboptimal beneficial effects on neurovascular function in people with HIV.
  • DOI:
    10.3389/fneur.2023.1240300
  • 发表时间:
    2023
  • 期刊:
  • 影响因子:
    3.4
  • 作者:
  • 通讯作者:
Sulforaphane prevents the reactivation of HIV-1 by suppressing NFκB signaling.
  • DOI:
    10.1016/j.jve.2023.100341
  • 发表时间:
    2023-09
  • 期刊:
  • 影响因子:
    5.5
  • 作者:
    Jamal I;Paudel A;Thompson L;Abdelmalek M;Khan IA;Singh VB
  • 通讯作者:
    Singh VB
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Vir Bahadur Singh其他文献

Vir Bahadur Singh的其他文献

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