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.

项目摘要 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，在这方面， 随着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型治疗靶点，以维持ART的收益。 HIV转录是病毒生命周期的重要组成部分，尚未成功靶向治疗。 我们最近发现，病毒必需蛋白达特与剪接因子3B亚基之间存在相互作用， 1（SF 3B 1）是HIV转录所需的。 该项目的主要目标是确定达特和SF 3B 1之间的相互作用在HIV中所起的作用 转录。长期目标是确定可以阻断这种相互作用的化合物，从而阻断HIV 复制的我们假设Tat介导的HIV转录需要达特与SF 3 B1相互作用， 当这种相互作用被阻断时，将抑制艾滋病毒的复制。我们的具体目标是： 目的1：确定HIV-1转录所需的Tat-SF 3B 1相互作用的分子结构域。我们 已经表明达特和SF 3 B1相互作用，并且这种相互作用需要达特的C-末端部分。然而，在这方面， 这种相互作用所需的达特或SF 3 B1的特定结构域是未知的。首先，我们将使用诱变， 沿着体外和体内蛋白结合研究，以缩小达特的残基和 相互作用所需的SF 3B 1。第二，我们将进行体内丙氨酸扫描，以更好地确定 两个结合域的相互作用表面。丙氨酸取代的影响将用以下方法定量： NanoLuc®二元技术（NanoBiT）裂解荧光素酶互补蛋白：蛋白相互作用系统。 我们还将在原代CD 4 + T细胞中进行Tat-SF 3B 1免疫沉淀和转录测定， 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。 目的2：明确SF 3B 1在Tat介导的HIV-1转录中的特异性作用。我们发现， SF 3B 1消除Tat介导的HIV转录，但SF 3B 1作用于HIV的确切机制 转录未知。SF 3 B1与PP 1（NIPP 1）的核抑制剂达特相互作用，并且也已知 甲基化HIV转录激活组蛋白H3 K36 me 3。因此，我们假设SF 3B 1可能控制 在原代CD 4 +T细胞、MDM和HIV潜伏模型中，HIV通过一种或多种这些途径转录。 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） 以定量SF 3B 1对达特/TAR相互作用的影响。其次，我们将确定SF 3B 1如何影响达特 甲基化以及H3 K36 me 3对HIV启动子的占据。最后，我们将确定Tat-SF 3B 1是否 相互作用在NIPP 1介导的HIV转录抑制中起作用。 这一工作对于进一步了解Tat介导的HIV转录和确定HIV基因的转录调控机制具有重要意义。 HIV的潜在治疗靶点。