Control of HIV replication by interactions between SF3B1 and Tat

通过 SF3B1 和 Tat 之间的相互作用控制 HIV 复制

• 批准号: 10468266
• 负责人: George Kyei
• 金额: $ 19.67万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-11 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10468266
• 关键词: Affect; Alanine; Amino Acids; Binding; Binding Proteins; Biological Assay; C-terminal; CD4 Positive T Lymphocytes; Cells; Chronic Disease; Complement; Complex; Data; Drug resistance; Electrophoretic Mobility Shift Assay; Engineering; Environment; Enzymes; Future; Genetic Transcription; Goals; HIV; HIV Infections; HIV therapy; HIV-1; Histones; Immunoprecipitation; In Vitro; Integrase Inhibitors; Life Cycle Stages; Luciferases; Mammalian Cell; Measures; Mediating; Methylation; Modeling; Mutagenesis; Neural Tube Defects; Nuclear; Pathway interactions; Physiological; Play; Proteins; RNA; RNA Polymerase II; RNA Splicing; Resistance; Role; Scanning; Signal Transduction; Surface; System; Technology; Testing; Therapeutic; Therapeutic Intervention; Viral; Viral Proteins; Weight Gain; Work; adverse drug reaction; antiretroviral therapy; base; chromatin immunoprecipitation; cyclin T1; experience; fight against; genetic manipulation; in vivo; inhibitor; interest; knock-down; luminescence; macrophage; molecular domain; monocyte; mutant; nanoluciferase; neuropsychiatric disorder; new therapeutic target; novel; pathogen; promoter; protein protein interaction; targeted treatment; tat Protein; therapeutic target

PROJECT SUMMARY Antiretroviral therapy (ART) has transformed HIV into a manageable non-fatal chronic disease. However, significant challenges remain with increasing drug resistance and adverse drug reactions. Therefore, there is an urgent need to identify novel therapeutic targets with unique mechanisms of action to sustain the gains of ART. HIV transcription represents a vital part of the viral life cycle that has not been successfully targeted for therapy. We recently discovered that an interaction between the essential viral protein Tat and splicing factor 3B subunit 1 (SF3B1) is required for HIV transcription. The primary goal of this project is to define the role played by the interaction between Tat and SF3B1 in HIV transcription. The long term objective is to identify compounds that can block this interaction and therefore HIV replication. We hypothesize that Tat-mediated HIV transcription requires Tat to interacts with SF3B1 and that this interaction, when blocked will inhibit HIV replication. Our specific aims are: Aim 1: Identify the molecular domains of Tat-SF3B1 interactions required for HIV-1 transcription. We have shown that Tat and SF3B1 interact, and that this interaction requires the C-terminal portion of Tat. However, the specific domains of Tat or SF3B1 required for this interaction are unknown. First, we will use mutagenesis, along with in-vitro and in-vivo protein binding studies to narrow down the residues of Tat and the domain of SF3B1 required for the interaction. Second, we will perform in-vivo alanine scanning to better define the interaction surfaces for the two binding domains. The effect of alanine substitutions will be quantified with the NanoLuc® Binary Technology (NanoBiT) split luciferase complementation protein: protein interaction system. We will also perform Tat-SF3B1 immunoprecipitations and transcription assays in primary CD4+ T cells and monocyte-derived macrophages (MDMs) to ascertain the physiological relevance of our findings. Aim 2: Define the specific role of SF3B1 in Tat-mediated HIV-1 transcription. We showed that depletion of SF3B1 abrogates Tat-mediated HIV transcription, but the exact mechanism for SF3B1 actions on HIV transcription is unknown. SF3B1 interacts with Tat, the nuclear inhibitor of PP1 (NIPP1), and is also known to methylate HIV transcription-activating histone H3K36me3. Therefore, we hypothesize that SF3B1 may control HIV transcription through one or more of these pathways in primary CD4+T cells, MDMs and HIV latency models. First, we will use RNA Immunoprecipitation (RNAIP) and fluorescent electrophoretic mobility shift assay (fEMSA) to quantify the effects of SF3B1 on Tat/TAR interactions. Second, we will determine how SF3B1 influences Tat methylation as well as the occupancy of the HIV promoter by H3K36me3. Finally we will determine if Tat-SF3B1 interactions plays a role in NIPP1-mediated inhibition of HIV transcription. This work will be highly significant to further the understanding of Tat-mediated HIV transcription and define a potential therapeutic target for HIV.

项目概要 抗逆转录病毒疗法（ART）已将艾滋病毒转变为一种可控制的非致命性慢性疾病。然而， 耐药性和药物不良反应的增加仍然存在重大挑战。因此，有一个 迫切需要确定具有独特作用机制的新治疗靶点，以维持 ART 的成果。 HIV转录是病毒生命周期的重要组成部分，但尚未成功地用于治疗。 我们最近发现必需病毒蛋白 Tat 和剪接因子 3B 亚基之间存在相互作用 HIV 转录需要 1 (SF3B1)。 该项目的主要目标是确定 Tat 和 SF3B1 之间的相互作用在 HIV 中所起的作用 转录。长期目标是找出可以阻断这种相互作用的化合物，从而阻断艾滋病毒 复制。我们假设 Tat 介导的 HIV 转录需要 Tat 与 SF3B1 相互作用，并且 这种相互作用一旦被阻断就会抑制艾滋病毒的复制。我们的具体目标是： 目标 1：确定 HIV-1 转录所需的 Tat-SF3B1 相互作用的分子结构域。我们 已经表明 Tat 和 SF3B1 相互作用，并且这种相互作用需要 Tat 的 C 端部分。然而， 这种相互作用所需的 Tat 或 SF3B1 的具体结构域尚不清楚。首先，我们将使用诱变， 以及体外和体内蛋白质结合研究，以缩小 Tat 的残基和 交互需要 SF3B1。其次，我们将进行体内丙氨酸扫描以更好地定义 两个结合域的相互作用表面。丙氨酸取代的影响将通过以下方式量化 NanoLuc® 二元技术 (NanoBiT) 分裂荧光素酶互补蛋白：蛋白质相互作用系统。 我们还将在原代 CD4+ T 细胞和 单核细胞衍生的巨噬细胞（MDM）以确定我们的研究结果的生理相关性。 目标 2：确定 SF3B1 在 Tat 介导的 HIV-1 转录中的具体作用。我们表明，耗尽 SF3B1 废除 Tat 介导的 HIV 转录，但 SF3B1 对 HIV 作用的确切机制 转录未知。 SF3B1 与 PP1 (NIPP1) 核抑制剂 Tat 相互作用，并且还已知 甲基化 HIV 转录激活组蛋白 H3K36me3。因此，我们假设SF3B1可能控制 HIV 转录通过原代 CD4+T 细胞、MDM 和 HIV 潜伏模型中的一种或多种途径进行。 首先，我们将使用 RNA 免疫沉淀 (RNAIP) 和荧光电泳迁移率变动分析 (fEMSA) 量化 SF3B1 对 Tat/TAR 相互作用的影响。其次，我们将确定 SF3B1 如何影响 Tat 甲基化以及 H3K36me3 对 HIV 启动子的占据。最后我们将确定Tat-SF3B1是否 相互作用在 NIPP1 介导的 HIV 转录抑制中发挥作用。 这项工作对于进一步了解 Tat 介导的 HIV 转录并定义一个 HIV的潜在治疗靶点。