Fate of Invisible U/A Base Pairs Within HIV DNA in Myeloid Phagocytic Cells

骨髓吞噬细胞中 HIV DNA 中看不见的 U/A 碱基对的命运

• 批准号: 9138025
• 负责人: JAMES T. STIVERS
• 金额: $ 40.15万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9138025
• 关键词: Acquired Immunodeficiency Syndrome; Adenine; Affect; Alveolar; Alveolar Macrophages; Anti-Inflammatory Agents; Anti-inflammatory; Base Excision Repairs; Base Pairing; Biochemical; Bone Marrow; Bronchial Lavages; CD4 Positive T Lymphocytes; CMV promoter; Cells; Characteristics; Chromatin; Code; Cytosine; DNA; DNA Fragmentation; DNA Repair; DNA Sequence; DNA-Binding Proteins; Deoxyuridine; Detection; Drug Targeting; Elements; Environment; Enzymes; Epigenetic Process; Excision; Gene Expression; Genetic Transcription; Genetic Variation; Genome; Goals; HIV; HIV Infections; HIV-1; Human; Immune; Immunology; In Vitro; Infection; Inflammatory; Intervention; Irrigation; Knowledge; Life; Lung; Methods; Mutagenesis; Mutation; Myelogenous; Myeloid Cells; Nuclear; Outcome; Pathway interactions; Patients; Phagocytes; Proviruses; RNA-Directed DNA Polymerase; Reading; Reporting; Rest; Reverse Transcription; Role; Route; Sampling; Scientist; System; T-Lymphocyte; Testing; Time; Tissues; Up-Regulation; Uracil; Viral; Virus; Virus Diseases; Work; base; cell type; combat; cytokine; enzyme activity; knock-down; macrophage; monocyte; novel; overexpression; peripheral blood; promoter; public health relevance; repair enzyme; repaired; restoration; small molecule; time use; uracil-DNA glycosylase; viral DNA; volunteer

 DESCRIPTION (provided by applicant): We have delineated the elements of a novel HIV-1 restriction/persistence mechanism that operates solely within the unique intracellular environment of bone marrow-derived monocytes (MCs) and monocyte-derived macrophages (MDMs), but not resting or activated T cells. The mechanism centers on the high concentration of dUTP in MCs and MDMs, which forces reverse transcriptase (RTase) to incorporate dUMP opposite to adenine during reverse transcription, leading to high levels of U/A base pairs ("uracilation"). Importantly, U/A pairs do not perturb the coding sequence and are invisible to standard DNA sequencing methods because U reads as T. However, U/A pairs are a substrate for the host uracil base excision repair (UBER) machinery, which modulates the outcome of HIV infection in these cells. Uracilation and UBER impact HIV infection of macrophages in diverse ways. First, potent pre- integration restriction occurs by uracil excision and viral DNA fragmentation. Proviruses that escape initial restriction contain U/A pairs that can persist, resulting in uracil- induced silencing of HIV-1 gene expression. Upon stimulation of infected MDMs with pro-inflammatory cytokines, otherwise stable uracilated proviruses become heavily mutagenized through error-prone UBER, suggesting that the M1/M2 polarization state of the macrophage can determine the fate viral uracils. Low levels of infectious virus can emerge from uracilated proviruses even after many weeks. In three aims we seek to (i) Elucidate the impact of the UBER pathway in HIV infection of macrophages and MCs. (ii) Determine the effects of U/A base pairs on HIV LTR-driven transcription in MDMs, and (iii) Use the time frame for uracil removal and its characteristic mutagenic signature to determine whether proviruses that have been previously detected in short-lived circulating MCs in HIV infected patients on cART originate from their contact with infected macrophages in various tissues. The elucidation of the role of host UBER in viral infections of macrophages and monocytes is expected to uncover new host drug targets for combating HIV establishment and persistence in these unique immune cells. This proposal brings together clinicians and basic scientists with broad and highly complementary backgrounds in immunology, HIV latency, uracil DNA repair and in obtaining alveolar macrophages from bronchial lavage of HIV-infected volunteers.

 描述（由申请人提供）：我们已经描述了一种新型 HIV-1 限制/持久性机制的要素，该机制仅在骨髓来源的单核细胞 (MC) 和单核细胞来源的巨噬细胞 (MDM) 的独特细胞内环境中起作用，但不该机制集中在 MC 和 MDM 中的高浓度 dUTP，这迫使逆转录酶 (RTase) 掺入与 dUMP 相反的物质。逆转录过程中腺嘌呤，导致高水平的 U/A 碱基对（“尿嘧啶化”）。重要的是，U/A 对不会干扰编码序列，并且对于标准 DNA 测序方法来说是不可见的，因为 U 读作 T。 /A 对是宿主尿嘧啶碱基切除修复 (UBER) 机制的底物，它通过多种方式调节这些细胞中 HIV 感染的结果。逃脱最初限制的原病毒含有可以持续存在的U/A对，导致尿嘧啶诱导的HIV-1基因表达沉默，在用促炎细胞因子刺激感染的MDM后，原本稳定的尿嘧啶原病毒变成。通过容易出错的 UBER 进行严重诱变，表明巨噬细胞的 M1/M2 极化状态可以决定病毒尿嘧啶的命运，从而可以产生低水平的传染性病毒。我们的三个目标是 (i) 阐明 UBER 途径对巨噬细胞和 MC 的 HIV 感染的影响 (ii) 确定 U/A 碱基对对 HIV LTR 驱动的转录的影响。 MDM，以及 (iii) 使用尿嘧啶去除的时间范围及其特征诱变特征来确定先前在接受 cART 的 HIV 感染患者的短命循环 MC 中检测到的原病毒是否源自阐明宿主 UBER 在巨噬细胞和单核细胞病毒感染中的作用有望发现新的宿主药物靶标，以对抗 HIV 在这些独特的免疫细胞中的建立和持续存在。以及在免疫学、HIV 潜伏期、尿嘧啶 DNA 修复以及从 HIV 感染志愿者的支气管灌洗中获取肺泡巨噬细胞方面具有广泛且高度互补背景的基础科学家。