Mechanism of Transcriptional Downregulation of HIV Gene Expression by U/A Base Pairs in Proviral DNA

前病毒 DNA 中 U/A 碱基对转录下调 HIV 基因表达的机制

• 批准号: 10053228
• 负责人: Anthony Gizzi
• 金额: $ 6.49万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-16 至 2021-06-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10053228
• 关键词: Address; Affect; Affinity; Alveolar Macrophages; Antibodies; Base Pairing; Binding; Binding Sites; Cell Line; Cells; Chromatin Structure; DNA; DNA Binding; DNA Polymerase II; DNA-Directed DNA Polymerase; Down-Regulation; Drug Targeting; Engineering; Enhancers; Excision; Fluorescence; Fluorescence Anisotropy; Frequencies; Gene Expression; Genetic Transcription; Genome; Goals; HIV; HIV Infections; HIV Transactivator Protein; HIV-1; Histones; Human; In Vitro; Infection; Interphase Cell; Knock-out; Long Terminal Repeats; Maps; Measurement; Measures; Methodology; Methods; Micrococcal Nuclease; Monitor; Mutagenesis; Mutation; Nature; Nucleosomes; Nucleotides; Outcome; Output; Polymerase; Positioning Attribute; Proviruses; RNA; RNA Polymerase II; RNA-Directed DNA Polymerase; Reporter; Reporting; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Site; Thymidine; Time; Transcription Elongation; Transcription Initiation; Transcription Initiation Site; Transfection; Uracil; Viral; Viral Genes; Virus Latency; antiretroviral therapy; base; cell type; dUTP pyrophosphatase; experimental study; flexibility; fluorophore; genomic RNA; in vivo; inducible gene expression; macrophage; monocyte; novel; prevent; promoter; transcription factor; uracil-DNA glycosylase; viral DNA; viral genomics

PROJECT SUMMARY/ABSTRACT Monocyte-derived macrophages (MDM) differentiated from primary human monocytes express high levels of the dNTP triphosphohydrolase SAMHD1 and undetectable dUTPase, leading to elevated ratios of dUTP/TTP in these non-dividing cells. This imbalanced dNTP pool has a profound effect on HIV infection by a mechanism involving incorporation of dUMP into viral DNA by reverse transcriptase, producing abundant U/A base pairs ("uracilation"). This is a unique aspect of HIV infection in this target cell type. Due to the low expression level of uracil DNA glycosylase in MDM, integrated uracilated proviruses persist for at least one month in vitro and can also be detected in monocytes and alveolar macrophages isolated from HIV infected people on long-term anti- retroviral therapy (ART). One emerging aspect of persistent proviral U/A pairs is their ability to decrease viral gene expression and induce transcriptional mutagenesis suggesting they may serve as a previously unrecognized mechanism of viral latency. In three aims, the broad goal of this proposal is to understand the effects of U/A base pairs on transcription factor (TF) occupancy, chromatin structure and RNA pol II activity with respect to HIV proviral DNA in MDM. In the first aim we will use a novel uracil sequencing method with single- nucleotide resolution to map U/A pairs within the 5'-long terminal repeat (LTR) promoter of HIV proviral DNA in MDM. This methodology is enabling because all other polymerase-based sequencing methods read uracil as thymidine. High-resolution mapping of the U/A pairs will establish which TF binding sites are modified during HIV infection of MDM and whether the RNA Pol II transcription initiation site also contains destabilizing U/A pairs. In a second aim, we will quantify the effects of U/A pairs on TF binding and RNA Pol II initiation and elongation in vitro and in human cells. In vitro TF DNA binding measurements will employ cognate binding sequences with site-specific T/A→U/A substitutions to quantify the effect of single- and multiple-site substitutions. Human cell studies will involve transfection of an eGFP reporter DNA containing site-specific U/A base pairs in the LTR region into an engineered cell line that is deficient in uracil excision and also expresses HIV transactivator protein. The relative levels of transcription from uracilated and reference (all-T) reporter constructs will be quantified by fluorescence and RT-PCR measurements. We will also perform ChIP experiments using TF specific antibodies to determine if the occupancy of TFs is affected by specific U/A pairs. The third aim will elucidate the effect of U/A pairs on nucleosome and TF occupancy and chromatin structure in infected MDM target cells. High-resolution nucleosome mapping by micrococcal nuclease (MNase) sensitivity will be used to determine differences in chromatin structure arising from U/A pairs in proviral DNA. These experiments will extend our understanding of the transcriptional effects of uracilation to the most relevant cell-type and explore the effects on chromatin structure that are relevant to infection of cells.

项目摘要/摘要 单核细胞衍生的巨噬细胞（MDM）与原代人单核细胞区分开 DNTP三磷酶SAMHD1和不可检测的DUTPase，导致DUTP/TTP的比率升高 这些非分散细胞。这种不平衡的DNTP池对通过机制对艾滋病毒感染产生了深远的影响 通过逆转录酶涉及掺入的并入掺入DNA，产生丰富的U/A碱基对 （“尿液”）。这是该靶细胞类型中HIV感染的独特方面。由于表达水平低 MDM中的尿嘧啶DNA糖基化酶，尿尿尿道病毒持续至少一个月的体外，可以 还可以在从艾滋病毒感染的人群中分离出的单核细胞和肺泡巨噬细胞中 逆转录病毒疗法（ART）。持续前病毒u/a对的一个新兴方面是它们降低病毒的能力 基因表达并诱导转录诱变表明它们可以用作先前 病毒潜伏期的未识别机制。在三个目标中，该提议的广泛目标是了解 U/A碱对对转录因子（TF）占用率，染色质结构和RNA POL II活性的影响 尊重MDM中的HIV前病毒DNA。在第一个目标中，我们将使用一种新型的尿嘧啶测序方法 核苷酸分辨率在5'长的末端重复启动子（LTR）中映射u/a对的HIV病毒前DNA MDM。这种方法之所以能够启用，是因为所有其他基于聚合酶的测序方法将尿嘧啶读为 胸苷。 U/A对的高分辨率映射将确定在HIV期间修改了哪些TF结合位点 MDM的感染以及RNA POL II转录启动位点是否包含U/A对的稳定。在 第二个目的，我们将量化U/A对对TF结合和RNA POL II启动和伸长的影响 体外和人类细胞。体外TF DNA结合测量结果将采用与 特定于位点的T/A→U/A取代，以量化单位和多站点取代的效果。人类细胞 研究将涉及转换LTR中包含位点特异性U/A碱基的EGFP报告基因DNA 区域进入工程细胞系，该细胞系在尿嘧啶切除术中具有明确的区域，也表达HIV反式激活器 蛋白质。尿液和参考（ALL-T）报告构建体的转录相对水平将是 通过荧光和RT-PCR测量进行量化。我们还将使用TF执行芯片实验 确定TFS占用率是否受特定U/A对的影响的特定抗体。第三个目标 阐明U/A对对感染MDM中核小体和TF占用率和染色质结构的影响 靶细胞。微分球菌核酸酶（MNase）敏感性的高分辨率核小体映射将用于 确定病毒DNA中U/A对产生的染色质结构的差异。这些实验会 将我们对尿液的转录效应的理解扩展到最相关的细胞类型并探索 与细胞感染相关的染色质结构的影响。