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.

项目概要/摘要 从原代人单核细胞分化而来的单核细胞衍生巨噬细胞 (MDM) 表达高水平的 dNTP 三磷酸水解酶 SAMHD1 和检测不到的 dUTPase，导致 dUTP/TTP 比率升高 这些不分裂的细胞通过某种机制对 HIV 感染产生深远的影响。 通过逆转录酶将 dUMP 掺入病毒 DNA，产生丰富的 U/A 碱基对 （“尿嘧啶”）这是该靶细胞类型中 HIV 感染的一个独特方面。 MDM 中的尿嘧啶 DNA 糖基化酶，整合的尿嘧啶原病毒在体外持续至少一个月，并且可以 在长期抗病毒治疗的 HIV 感染者中分离出的单核细胞和肺泡巨噬细胞中也可检测到 逆转录病毒疗法 (ART) 持久性原病毒 U/A 对的一个新兴方面是它们减少病毒的能力。 基因表达并诱导转录突变，表明它们可能作为先前的 该提案的主要目标是了解未知的病毒潜伏机制。 U/A 碱基对对转录因子 (TF) 占用、染色质结构和 RNA pol II 活性的影响 关于 MDM 中的 HIV 前病毒 DNA，我们将使用一种新颖的单尿嘧啶测序方法。 核苷酸分辨率以绘制 HIV 前病毒 DNA 5' 长末端重复 (LTR) 启动子内的 U/A 对 MDM。这种方法是可行的，因为所有其他基于聚合酶的测序方法都将尿嘧啶读取为 U/A 对的高分辨率图谱将确定哪些 TF 结合位点在 HIV 过程中被修饰。 MDM 感染以及 RNA Pol II 转录起始位点是否也包含不稳定的 U/A In 对。 第二个目标是，我们将量化 U/A 对对 TF 结合以及 RNA Pol II 起始和延伸的影响 体外和人类细胞中的 TF DNA 结合测量将采用同源结合序列 位点特异性 T/A→U/A 替换，以量化单位点和多位点替换的效果。 研究将涉及转染 LTR 中包含位点特异性 U/A 碱基对的 eGFP 报告 DNA 区域转化为尿嘧啶切除缺陷但也表达 HIV 反式激活因子的工程细胞系 尿嘧啶化和参考（全 T）报告基因构建体的转录相对水平为 我们还将使用 TF 进行 ChIP 实验。 第三个目标是使用特异性抗体来确定 TF 的占用是否受到特定 U/A 对的影响。 阐明 U/A 对对受感染 MDM 中核小体和 TF 占据以及染色质结构的影响 通过微球菌核酸酶 (MNase) 敏感性进行的高分辨率核小体图谱将用于 确定原病毒 DNA 中 U/A 对产生的染色质结构差异。 将我们对尿嘧啶转录效应的理解扩展到最相关的细胞类型并探索 对与细胞感染相关的染色质结构的影响。