Identification and characterization of chromatin regulators of HIV-1 latency

HIV-1 潜伏期染色质调节因子的鉴定和表征

• 批准号: 10458119
• 负责人: Susana T Valente
• 金额: $ 92.18万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10458119
• 关键词: ATP phosphohydrolase; Affinity Chromatography; Anti-Retroviral Agents; Architecture; BLT mice; Bacterial Artificial Chromosomes; Binding; Biochemical; Biological; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Cell model; Cells; ChIP-seq; Chromatin; Chromatin Remodeling Factor; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Data; Epigenetic Process; Family; Feedback; Gene Expression; Genes; Genetic Transcription; Genome; Goals; HIV; HIV Genome; HIV-1; Human; ISWI; Individual; Interruption; Investigation; Libraries; Link; Mediating; Modeling; Molecular; Mus; Nature; NuRD complex; Nucleosomes; Patients; Pharmacology; Phase; Phenotype; Positioning Attribute; Production; Property; RNA interference screen; Reactive Inhibition; Reproducibility; Residual state; Resistance; Resolution; Retroviral Vector; Sampling; Signal Transduction; Specificity; System; T memory cell; Techniques; Testing; Therapeutic; Tissues; Transcript; Transcription Initiation Site; Viral; Viremia; Virus; Virus Diseases; Writing; base; chromatin modification; chromatin remodeling; combinatorial; cortistatin; experimental study; follow-up; inhibitor; insight; integration site; knock-down; mouse model; novel; particle; prevent; promoter; recruit; screening; small hairpin RNA; tat Protein; transcriptome sequencing; viral RNA; viral rebound

Abstract After HIV integration of the proviral DNA into the host genome, the virus can remain latent or activate transcription. The viral Tat protein, which enhances transcript elongation from the HIV-1 promoter, is the switch between these two states. Since Tat resides under the control of the same promoter, it enhances its own transcription via a positive feedback loop. We identified didehydro-Cortistatin A (dCA) as a very potent inhibitor of Tat (1, 2). In human CD4 +T cells isolated from aviremic individuals, combining dCA with ART accelerates HIV-1 suppression and prevents viral rebound during treatment interruption, as the HIV-1 promoter remains epigenetically repressed. HIV-1 transcriptional inhibitors have the unique property of reducing particle production from infected cells. dCA is the proof-of-concept that this novel class of molecules is amenable to block-and-lock functional cure approaches, which aim at reducing residual viremia during ART and limit viral rebound. It is thus important to understand the mechanisms that explain not only dCA's inhibition of reactivation, but also mechanisms regulating HIV-1 latency in CD4+T memory T cells in general, to expand on “block-and-lock” approaches, and explore alternative options for retroviral suppression. There are approximately 320 human chromatin regulators, which “write”, “erase”, or “read” chromatin modifications, or remodel nucleosome topology. Specificity in gene expression derives from the combinatorial nature of chromatin modifications, and assembly of related chromatin regulator subunits. The rationale for this proposal is that factors that establish HIV-1 latency are important for viral reactivation, and that by identifying and inhibiting them, a “locked” state of silencing that is exceedingly resistant to reactivation can be achieved. We propose to combine a comprehensive high-resolution mapping of the nucleosome organization and positioning of chromatin remodeling complexes at the HIV promoter during HIV latency, with a robust pooled shRNAs screening approach to interrogate all chromatin regulatory factors in parallel during a single experiment. Primary and secondary screens will be performed in a newly developed primary cell system that captures bona fide HIV-1 latency, and departs from CD4+T cells from successfully treated HIV infected donors. During the R61 phase of the project we will be able to correlate high-resolution nucleosome architecture data with their binding to all chromatin remodeling machine families and develop a comprehensive picture of the signals and factors that drive chromatin activity at the HIV-1 genome during latency. This data will support robust hypotheses and targets to test in detail during the R33 phase. We anticipate that from these candidates, we can infer how HIV-latency is controlled and develop rational therapeutic approaches to modulate HIV latency.

摘要 在HIV将前病毒DNA整合到宿主基因组中之后，病毒可以保持潜伏或激活 转录。病毒达特蛋白是一个开关，它能增强HIV-1启动子的转录延长 在这两个国家之间。由于达特位于同一启动子的控制下，因此它增强了自身的转录活性。 通过正反馈循环转录。我们鉴定了双脱氢皮质抑素A（dCA）作为一种非常有效的 达特（1，2）抑制剂。在从病毒血症个体分离的人CD 4 +T细胞中，将dCA与ART组合 作为HIV-1促进剂，加速HIV-1抑制并防止治疗中断期间病毒反弹 在表观遗传学上仍然受到抑制。HIV-1转录抑制剂具有独特的性质， 从受感染的细胞中产生。dCA是这类新型分子的概念验证， 阻断和锁定功能性治愈方法，旨在减少ART期间的残留病毒血症， 病毒反弹因此，重要的是要理解不仅可以解释dCA抑制 重新激活，而且还调节HIV-1在CD 4 +T记忆T细胞中潜伏期的机制，以扩展 “封锁和封锁”方法，并探索抑制逆转录病毒的替代办法。 人类染色质调节因子大约有320种，可以“写入”、“擦除”或“读取”染色质 修饰或重塑核小体拓扑结构。基因表达的特异性来源于组合 染色质修饰的性质和相关染色质调节子亚基的组装。这样做的理由 建议是，建立HIV-1潜伏期的因素对病毒的重新激活很重要， 并且抑制它们，可以实现对再激活具有极大抵抗力的沉默的“锁定”状态。 我们建议联合收割机将核小体的全面高分辨率绘图 HIV感染过程中染色质重塑复合物在HIV启动子上的组织和定位 潜伏期，使用稳健的混合shRNA筛选方法来询问所有染色质调节因子 在同一个实验中。初级和次级筛选将在新开发的 原代细胞系统，捕获真正的HIV-1潜伏期，并从CD 4 +T细胞成功地 接受过治疗的艾滋病毒感染者。 在项目的R61阶段，我们将能够将高分辨率的核小体 架构数据与它们绑定到所有染色质重塑机器家族，并开发一个全面的 在潜伏期期间驱动HIV-1基因组染色质活性的信号和因子的图片。该数据 将支持在R33阶段进行详细测试的可靠假设和目标。我们预计，从这些 候选人，我们可以推断如何控制艾滋病毒潜伏期，并制定合理的治疗方法， 调节HIV潜伏期。