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

项目摘要/摘要 从原代人单核细胞分化而来的单核细胞来源的巨噬细胞(MDM)表达高水平的 DNTP三磷酸水解酶SAMHD1和未检测到的dUTP酶导致dUTP/TTP比值升高 这些未分裂的细胞。这种不平衡的dNTP池通过一种机制对艾滋病毒感染产生深远影响 通过逆转录酶将DUMP整合到病毒DNA中，产生丰富的U/A碱基对 (“尿嘧啶”)。这是这种靶细胞中HIV感染的一个独特方面。由于表达水平较低， MDM中的尿嘧啶DNA糖基酶，整合的尿嘧啶前病毒在体外至少持续一个月，并可以 也可在单核细胞和肺泡巨噬细胞中检测到分离自HIV感染者的长期抗病毒治疗 逆转录病毒疗法(ART)。持续性前病毒U/A对的一个新出现的方面是它们降低病毒的能力 基因表达和诱导转录突变提示它们可能作为先前的 未知的病毒潜伏机制。在三个目标中，本提案的总体目标是理解 U/A碱基对对转录因子(TF)占有率、染色质结构和RNA PolII活性的影响 MDM患者对HIV前病毒DNA的尊重。在第一个目标中，我们将使用一种新的尿嘧啶测序方法，该方法具有单一的- 人类免疫缺陷病毒前病毒DNA 5‘-末端重复序列启动子内U/A对的核苷酸解析 MDM。这种方法之所以可行，是因为所有其他基于聚合酶的测序方法都将尿嘧啶读作 胸苷。U/A对的高分辨率作图将确定哪些Tf结合位点在HIV期间被修改 MDM的感染以及RNA POL II转录起始点是否也包含不稳定的U/A对。在……里面 第二个目标，我们将量化U/A对Tf结合和RNA Pol II启动和延伸的影响 在体外和在人类细胞中。在体外，Tf DNA结合测量将使用同源结合序列 站点特定的T/A→U/A替换，以量化单站点和多站点替换的影响。人体细胞 研究将包括将含有位点特异性U/A碱基对的EGFP报告DNA导入LTR 区域转化为缺乏尿嘧啶切除并表达HIV反式激活因子的工程化细胞系 蛋白。来自uracilated和Reference(ALL-T)报告构建体的相对转录水平将为 通过荧光和RT-PCR测量进行定量。我们还将使用TF进行芯片实验 特异性抗体，以确定TF的占有率是否受特定U/A对的影响。第三个目标将是 阐明U/A对MDM感染后核小体、转铁蛋白占有率和染色质结构的影响 目标单元格。微球菌核酸酶(MNase)敏感性的高分辨率核小体图谱将用于 确定前病毒DNA中U/A对引起的染色质结构的差异。这些实验将 将我们对尿嘧啶作用转录效应的理解扩展到最相关的细胞类型，并探索 与细胞感染有关的染色质结构的影响。

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.