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：
Vir Bahadur Singh
金额：
$ 49.53万
依托单位：
ALBANY COLLEGE OF PHARMACY
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

来源：
https://reporter.nih.gov/project-details/10484480
关键词：
Address Area BRF1 gene Biochemical Biological Assay Biological Phenomena CD4 Positive T Lymphocytes Cartoons Cell Death Cell Line Cell model Cells Chromatin Clinical Complex DNA DNA Polymerase II DNA Polymerase III DNA-Directed RNA Polymerase Dose Ensure Epigenetic Process Exhibits Future Gene Expression Regulation Genes Genetic Genetic Transcription Genomics HIV HIV Genome HIV Infections HIV-1 Heterogeneity Individual Infection Interferons Intervention Investigation Knock-out Learning Long Terminal Repeats Measures Mediating Memory Molecular Molecular Virology Outcome Patients Play RNA Regulator Genes Reporting Research Rest Role Sampling Science Shock Signal Transduction Site T-Lymphocyte Techniques Testing Time Toxic effect Training Tretinoin Untranslated RNA Viral Viral Cytopathogenic Effect Viral Genes Viral reservoir Virus Latency Work Yeasts cell type chromatin immunoprecipitation design experimental study inhibitor innovation integration site knock-down liposarcoma monocyte multidisciplinary novel novel strategies overexpression promoter purge selective expression therapeutic target transcriptome sequencing undergraduate student university student

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.

抽象的静息 CD4+ T 细胞在休眠期间携带大部分潜在的人类免疫缺陷病毒 (HIV) 由于涉及到感染，消除这种储存库是一项极具挑战性的任务。因此，目前可能没有一种机制可以调节艾滋病毒潜伏期。有前途的潜伏期逆转剂（LRA）能够减少潜伏原病毒的大小因此，迫切需要寻找新的机制和治疗靶点。可以靶向清除异质潜在储存库。似乎是一个主调节器，具有通过中介调节延迟的尚未开发的潜力不同的机制，例如 i) 调节邻近 RNA Pol II 基因的表达启动子和 ii) 具有调节表达潜力的新型非编码 RNA 的转录我们的初步研究表明 Pol III 转录富集。潜伏细胞中的非编码RNA，即散布在Alu之间的7SK、21A和BC200 这与 HIV 潜伏期高度相关，因为发现 HIV 基因组优先重复。经测试，使用 RNA Pol III 抑制剂 ML60218 进行整合潜伏细胞系 J89GFP 和 THP89GFP 前所未有的重新激活（高达 90%）此外，我们特别观察到高度的细胞死亡。由于病毒细胞病变效应，HIV 感染细胞中的病毒得以存活，而未感染的细胞则保持存活即使在非常高浓度的 RNA Pol III 抑制剂（100 μM）下，这些令人兴奋的发现和将利用确凿的报告来检验 RNA Pol III 发挥关键作用的假设在建立 HIV 潜伏期和靶向 RNA 中间新型效应子中的作用 Pol III 驱动机制可能会增强治疗策略的功效。这项研究存在分歧。分为两个具体目标，重点是研究直接驱动的 RNA Pol III（由基因组占据）和间接（通过 ncRNA）机制可能调节 HIV 潜伏期。 RNA Pol III 抑制/敲低的效果将在离体培养的原代 CD4+ T 细胞模型中进行测试。在目标 1.2 中，我们将进一步研究附近的 RNA Pol III 的物理存在是否可以改变 HIV 5´ LTR 处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。通过将 RNA-seq 与 RNA Pol III ChIP-Seq 结合使用，RNA 参与潜伏期。最后，将对选定的非编码 RNA 单独或在其中进行基因敲低研究组合来研究它们在促进 HIV 5´ LTR 抑制染色质状态中的作用。这项研究具有高度创新性，因为我们的目标是识别新型表观遗传调节剂同步瞄准克服与艾滋病毒休克和杀灭策略相关的挑战成功完成这项研究将提供可能的关键机制信息。解决潜在储层之间的非均质性。

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.

人类免疫缺陷病毒-1 Tat 通过下调 Sonic Hedgehog 信号传导发挥其神经毒性作用。

DOI：
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

Sulforaphane prevents the reactivation of HIV-1 by suppressing NFκB signaling.

萝卜硫素通过抑制 NFκB 信号传导来防止 HIV-1 的重新激活。

DOI：
发表时间：
2023-09
期刊：
影响因子：
0
作者：
Jamal, Imran;Paudel, Anisha;Thompson, Landon;Abdelmalek, Michel;Khan, Irfan A;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.

联合抗逆转录病毒治疗的开始对艾滋病毒患者的神经血管功能产生次优的有益效果。

DOI：
发表时间：
2023
期刊：
影响因子：
0
作者：
Singh, Meera V;Uddin, Md Nasir;Singh, Vir B;Peterson, Angelique N;Murray, Kyle D;Zhuang, Yuchuan;Tyrell, Alicia;Wang, Lu;Tivarus, Madalina E;Zhong, Jianhui;Qiu, Xing;Schifitto, Giovanni
通讯作者：
Schifitto, Giovanni