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（由适用提供）：我们已经描述了一种新型的HIV-1约束/持久性机制的元素，该机制仅在骨髓衍生的单核细胞（MCS）和单核细胞衍生的巨噬细胞（MDMS）（MDMS）的独特细胞内环境中运行，但不静止或激活的T细胞。该机制集中在MCS和MDM中的DUTP高浓度上，它们迫使逆转录酶（RTase）在逆转录过程中掺入与腺嘌呤相对的倾倒，从而导致高水平的U/A碱基对（“尿液”）。重要的是，u/a对不会扰动编码顺序，并且是标准DNA测序方法不可见的，因为U读取为T。但是，U/A对是宿主Uracil碱基惊喜（UBER）机械（UBER）机械的底物，这调节了这些细胞中HIV感染的结果。尿液和Uber影响巨噬细胞的HIV感染以潜水员的方式。首先，潜在的预一体化限制是由于尿嘧啶惊喜和病毒DNA碎片而发生的。逃脱初始限制的原病毒包含可以持续存在的U/A对，导致尿嘧啶诱导的HIV-1基因表达沉默。通过促炎细胞因子刺激感染的MDM，否则稳定的尿液病毒通过容易出错的Uber诱发了大量诱变，这表明巨噬细胞的M1/M2极化状态可以确定脂肪病毒尿尿症。即使经过几周，也可能会从尿液病毒病毒中出现低水平的感染性病毒。在三个目标中，我们寻求（i）阐明Uber途径在巨噬细胞和MC的HIV感染中的影响。 （ii）确定U/A碱基对MDMS中HIV LTR驱动的转录的影响，（iii）使用时间范围来清除尿嘧啶及其特征性的诱变签名，以确定是否在HIV循环的MCS中发现了先前在HIV循环的MC中检测到的预科病毒，这是HIV感染的患者在CART中源自其与受感染的莫克果的接触。阐明宿主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，以打击这些独特的免疫细胞中HIV的持续性和持久性。该提案汇集了具有免疫学，艾滋病毒潜伏期，尿嘧啶DNA修复以及从HIV感染的志愿者支气管灌洗中获得肺泡巨噬细胞的广泛而高度完整的临床医生和基础科学家。