Control of HIV replication by interactions between SF3B1 and Tat

项目总结 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病。然而， 随着耐药性的增加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的治疗靶点，以维持抗逆转录病毒治疗的成果。 艾滋病毒转录是病毒生命周期中的一个重要部分，但尚未成功用于治疗。 我们最近发现，必需的病毒蛋白TAT和剪接因子3B亚单位之间的相互作用 1(SF3B1)是HIV转录所必需的。 这个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 抄写。长期目标是确定能够阻止这种相互作用的化合物，从而阻止艾滋病毒 复制。我们假设TAT介导的HIV转录需要TAT与SF3B1相互作用，并且 当这种相互作用被阻止时，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-SF3B1相互作用的分子结构域。我们 已经证明TAT和SF3B1是相互作用的，这种相互作用需要TAT的C末端部分。然而， 这种相互作用所需的TAT或SF3B1的特定结构域是未知的。首先，我们将使用突变技术， 以及体外和体内蛋白质结合研究，以缩小TAT的残留和TAT的结构域 交互所需的SF3B1。其次，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面。丙氨酸取代的影响将用 NanoLuc®二元技术(Nanobit)拆分荧光素酶互补蛋白：蛋白质相互作用系统。 我们还将在原代CD4T细胞中进行TAT-SF3B1免疫沉淀和转录检测 单核细胞来源的巨噬细胞(MDM)，以确定我们发现的生理相关性。 目的2：明确SF3B1在TAT介导的HIV-1转录中的特殊作用。我们发现了耗尽的 SF3B1取消了TAT介导的HIV转录，但SF3B1对HIV作用的确切机制 转录情况尚不清楚。SF3B1与PP1(NIPP1)的核抑制物TAT相互作用，也是已知的 甲基化HIV转录激活组蛋白H3K36me3。因此，我们假设SF3B1可能控制着 在原代CD4T细胞、MDM和HIV潜伏期模型中，HIV通过一个或多个上述途径转录。 首先，我们将使用RNA免疫沉淀(RNAIP)和荧光电泳迁移率改变分析(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启动子的占位。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。 这项工作将对进一步了解TAT介导的HIV转录并确定 艾滋病毒的潜在治疗靶点。

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.