Host factors regulating HIV latency and reactivation

调节HIV潜伏期和再激活的宿主因素

• 批准号: 10516096
• 负责人: Susana T Valente
• 金额: $ 48.3万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-11-01 至 2026-10-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10516096
• 关键词: Acceleration; Affect; Affinity Chromatography; Automobile Driving; Binding; Binding Proteins; Biological; CD4 Positive T Lymphocytes; Cell model; Cells; Cellular Metabolic Process; ChIP-seq; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Dependence; ERCC3 gene; Elements; Environment; Epigenetic Process; Generic Drugs; Genes; Genetic Transcription; Goals; HIV; HIV Genome; HIV-1; Heterochromatin; Impairment; In Vitro; Individual; Integration Host Factors; Interruption; Knock-out; Knowledge; Link; Mass Spectrum Analysis; Mediating; Messenger RNA; Modification; Molecular; Molecular Conformation; Nucleosomes; Pathway interactions; Pharmaceutical Preparations; Positioning Attribute; Post-Translational Protein Processing; Productivity; Proliferating; Proteins; Proviruses; RNA Polymerase II; RNA Splicing; Reporting; Reproducibility; Residual state; Role; Spironolactone; Testing; Transactivation; Transcription Coactivator; Translational Protein Modification; Viral; Viral Proteins; Viremia; acrosome stabilizing factor; antiretroviral therapy; cortistatin; detection limit; epigenetic silencing; follow-up; in vivo; in vivo Model; inhibitor; insight; knock-down; novel; overexpression; prohibitin; promoter; protein complex; reactivation from latency; recruit; small hairpin RNA; small molecule; targeted agent; transcription factor; transcription factor TFIIH; transcriptomics; viral rebound

Abstract Our central premise is that HIV transcriptional inhibitors can be used as latency promoting agents (LPAs) in block-and-lock functional cure approaches, aimed at reducing residual viremia during antiretroviral therapy (ART) and limiting viral rebound upon treatment interruption (TI). This hypothesis came about with the discovery of the Tat inhibitor, didehydro-Cortistatin A (dCA). In in vitro and in vivo models of HIV latency combining ART with dCA accelerated HIV-1 suppression to below the limit of detection and blocked viral rebound upon TI or stimulation with latency reversing agents (LRAs). The transcriptional shutdown by dCA resulted in the heterochromatinization and loss of RNAPII at the HIV promoter. We recently reported on another LPA, the generic drug Spironolactone (SP). Interestingly this drug degrades the host XPB subunit of the general transcription factor TFIIH inhibiting HIV transcription in primary cells from infected individuals and blocking viral reactivation from latency without affecting cellular transcriptomics7. This study is important because it highlights that a host factor can be targeted to silence HIV without affecting cellular viability. In sum, the more knowledge we gain on the interconnectivity between Tat, transcription factors regulatory mechanisms and how these affect nucleosomes positioning around the HIV promoter, the more we can leverage transcriptional regulators as antiviral targets. To identify novel host factors regulating HIV transcription we used a chromatin affinity purification approach together with mass spectrometry (ChAP-MS). We identified p32 (ASF/SF2 splicing factor-associated protein) and FUBP3 (Far upstream element binding protein 3) in productively infected ART treated promoters but not in dCA-treated where HIV was silenced; and PHB2 (prohibitin-2) was enriched in dCA treated promoters but reduced in ART only treated ones. These results suggest that p32 and FUBP3 are HIV transcriptional activators while PHB2 is a HIV silencing factor. Preliminary studies confirmed their transcriptional activity on HIV. Here we propose a detailed molecular mechanistic study on how p32 and FUBP3 enhance and how PHB2 inhibits HIV transcription. Understanding their role in pre-initiation complex (PIC) formation, initiation, elongation and nucleosome organization at the latent HIV promoter will be key for developing successful strategies for durable HIV silencing. Strong effects of one or more of these factors will prompt the search for small-molecule modulators. Our overarching goal is to durably silence latent HIV proviruses by driving each into stable heterochromatin where they will remain “locked.” We propose the following aims: Aim 1: Define the roles of p32, FUBP3 and PHB2 in HIV transcription and chromatin structure. Aim 2: Identify protein complexes associated with p32, FUBP3 and PHB2 and study interaction with Tat. Aim 3: Investigate the “block-and-lock” approach using single or combinations of factors and their dependence on Tat/TAR circuitry.

抽象的 我们的中心前提是 HIV 转录抑制剂可以用作潜伏期促进剂 (LPA) 阻断和锁定功能性治愈方法，旨在减少抗逆转录病毒治疗 (ART) 期间的残留病毒血症 并限制治疗中断（TI）时的病毒反弹。这个假说是随着发现 Tat 抑制剂，二脱氢皮质抑素 A (dCA)。结合 ART 和 HIV 潜伏期的体外和体内模型 dCA 加速 HIV-1 抑制至检测限以下，并阻止病毒在 TI 或 潜伏期逆转剂（LRA）刺激。 dCA 的转录关闭导致 HIV 启动子处的异染色质化和 RNAPII 丢失。我们最近报道了另一个 LPA， 仿制药螺内酯（SP）。有趣的是，这种药物会降解一般宿主的 XPB 亚基。 转录因子 TFIIH 抑制受感染个体原代细胞中的 HIV 转录并阻断病毒 从潜伏期重新激活而不影响细胞转录组学7。这项研究很重要，因为它强调了 可以靶向宿主因子来沉默 HIV，而不影响细胞活力。总而言之，知识越多 我们了解了 Tat、转录因子调节机制之间的相互联系以及它们如何影响 核小体位于 HIV 启动子周围，我们就越能利用转录调节因子 抗病毒靶点。 为了确定调节 HIV 转录的新宿主因子，我们使用了染色质亲和纯化方法 与质谱 (ChAP-MS) 一起使用。我们鉴定出 p32（ASF/SF2 剪接因子相关蛋白） 和 FUBP3（远上游元件结合蛋白 3）存在于有效感染的 ART 处理的启动子中，但不在 经过 dCA 治疗，HIV 被抑制； PHB2 (prohibitin-2) 在 dCA 处理的启动子中富集，但 仅接受 ART 治疗的患者减少。这些结果表明 p32 和 FUBP3 是 HIV 转录因子 激活剂，而 PHB2 是 HIV 沉默因子。初步研究证实了它们的转录活性 艾滋病病毒。在这里，我们提出了关于 p32 和 FUBP3 如何增强以及 PHB2 如何增强的详细分子机制研究 抑制HIV转录。了解它们在起始复合物 (PIC) 形成、起始、延伸中的作用 潜在 HIV 启动子的核小体组织将是制定成功策略的关键 持久的艾滋病毒沉默。这些因素中的一个或多个的强烈影响将促使人们寻找小分子 调制器。我们的首要目标是通过使潜伏的艾滋病毒原病毒进入稳定状态来持久地沉默 异染色质将保持“锁定”状态。我们提出以下目标： 目标 1：定义 p32、FUBP3 和 PHB2 在 HIV 转录和染色质结构中的作用。 目标 2：鉴定与 p32、FUBP3 和 PHB2 相关的蛋白质复合物并研究与 Tat 的相互作用。 目标 3：使用单个因素或因素组合及其依赖性来研究“封锁和锁定”方法 在 Tat/TAR 电路上。