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: 10427641
• 负责人: Susana T Valente
• 金额: $ 46.25万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-22 至 2021-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10427641
• 关键词: Affect; Affinity Chromatography; Antiviral Agents; Automobile Driving; Binding; Binding Proteins; Biological; Biological Assay; Cells; ChIP-seq; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Dependence; ERCC3 gene; Elements; Environment; Epigenetic Process; FUBP3 gene; Generic Drugs; Genes; Genetic Transcription; Goals; HIV; HIV Genome; HIV-1; Heterochromatin; Impairment; In Vitro; Individual; Integration Host Factors; Interruption; Investigation; Knowledge; Link; Mass Spectrum Analysis; Modeling; Molecular; Molecular Conformation; Nucleosomes; Pathway interactions; Pharmaceutical Preparations; Positioning Attribute; Post-Translational Protein Processing; Proteins; Proviruses; RNA Polymerase II; RNA Splicing; Reporting; Reproducibility; Residual state; Role; Spironolactone; Sum; Testing; Transactivation; Transcription Coactivator; Transcription Initiation Site; Viral; Viral Proteins; Viremia; acrosome stabilizing factor; antiretroviral therapy; base; cortistatin; detection limit; epigenetic silencing; follow-up; in vivo; in vivo Model; inhibitor/antagonist; insight; knock-down; novel; overexpression; prohibitin; promoter; protein complex; reactivation from latency; recruit; small hairpin RNA; small molecule; tat Protein; transcription factor; transcription factor TFIIH; transcriptome sequencing; viral rebound

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)2,3. 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)4. The transcriptional shutdown by dCA resulted in the heterochromatinization and loss of RNAPII at the HIV promoter5. We recently reported on another LPA, the generic drug, Spironolactone (SP). Interestingly this drug degrades the host XPB subunit of the general transcription factor TFIIH6 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 assay together with mass spectrometry (ChAP-MS)8. 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: 1) Define the roles of p32, FUBP3 and PHB2 in HIV transcription and chromatin structure. 2) Identify protein complexes associated with p32, FUBP3 and PHB2. 3) Investigate p32, FUBP3 and PHB2 dependence on the Tat/TAR circuitry.

我们的主要前提是，HIV转录抑制剂可以用作促进剂（LPA） 阻塞功能治愈方法，旨在减少抗逆转录病毒治疗期间残留病毒血症（ART） 并在治疗中断（TI）时限制病毒反弹。这个假设是出于发现 TAT抑制剂，Didehydro-Cortistin A（DCA）2,3。艾滋病毒潜伏期的体外和体内模型，将艺术与 DCA加速HIV-1抑制至低于检测极限，并在Ti或 刺激潜伏期逆转剂（LRAS）4。 DCA的转录关闭导致 HIV启动子5的异染色和RNAPII丢失。我们最近报告了另一个LPA 通用药物，螺内酯（SP）。有趣的是，这种药物降低了一般的宿主XPB亚基 转录因子TFIIH6抑制感染个体的原代细胞中HIV转录并阻止病毒 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，因为它突出了 可以将宿主因子靶向沉默HIV而不会影响细胞活力。总而言之 我们获得了TAT之间的互连性，转录因子调节机制以及这些影响如何影响 在艾滋病毒启动子周围定位的核小体，我们越能利用转录调节因子作为 抗病毒靶标。 为了识别调查HIV转录的新型宿主因素，我们使用了染色质亲和力纯化测定法 与质谱法（第MS）8一起。我们确定了p32（ASF/SF2剪接因子相关蛋白） 在有效感染的ART处理的启动子中，fubp3（远上游元件结合蛋白3），但不在 DCA治疗的艾滋病毒被沉默的地方；在DCA处理的启动子中富含PHB2（禁止素-2），但 在艺术中​​减少了。这些结果表明p32和fubp3是HIV转录的 激活剂虽然PHB2是HIV沉默因素。初步研究证实了它们对 艾滋病病毒。在这里，我们提出了一项详细的分子机械研究，介绍了p32和fubp3如何增强以及PHB2的方式 抑制HIV转录。了解它们在生成复合物（PIC）形成，启动，伸长率中的作用 潜伏艾滋病毒促进者的核小体组织将是制定成功策略的关键 耐用的HIV沉默。其中一个或多个因素的强烈影响将促使人们寻找小分子 调节器。我们的总体目标是通过将每个人都带入稳定来使持久的沉默潜在的艾滋病毒病毒 它们将保持“锁定”。 我们提出以下目标： 1）定义p32，fubp3和phb2在HIV转录和染色质结构中的作用。 2）确定与p32，fubp3和PHB2相关的蛋白质复合物。 3）研究P32，FUBP3和PHB2对TAT/TAR电路的依赖性。