Multifaceted interactions between lentiviral Vif and host molecules for viral infectivity enhancement

项目描述 APOBEC 3（A3）蛋白家族是细胞胞苷脱氨酶，其抑制人类免疫应答。 免疫缺陷病毒1型（HIV-1）感染通过病毒逆转录和物理 阻断逆转录。为了逃避这种宿主防御机制，HIV-1表达了病毒粒子的感染性， 因子（Vif），其劫持细胞E3泛素连接酶复合物并靶向A3蛋白（A3 F/G/H/D）， 蛋白酶体介导的降解。除了降解A3，HIV-1 Vif还通过以下方式引起G2细胞周期阻滞： 靶向多种蛋白磷酸酶2A（PP 2A）调节剂（PPP 2 R5蛋白）进行降解。增加了 由于Vif-宿主蛋白相互作用的复杂性，HIV-1 Vif利用转录因子CBFβ作为非典型的 辅因子，而梅迪-维斯纳病毒（MVV）Vif替代脯氨酰异构酶亲环素A（CypA）。我们的目标 是建立慢病毒Vif分子多方面活动的生物化学和结构原理 通过泛素-蛋白酶体途径募集细胞因子降解宿主蛋白质。为了实现我们的目标， 我们将结合使用生物化学、生物物理学、结构生物学和细胞功能技术。到 建立A3蛋白被HIV-1 Vif（Aim 1）靶向的机制，我们将确定高水平的 A3-Vif-E3相互作用复合物的解析结构，通过结构引导的 体外和体内诱变实验，并询问Vif/A3/E3连接酶的分子决定簇 组装和激活。此外，我们还将研究Vif抑制的非降解依赖模式， A3脱氨基和抗病毒活性。为了更好地理解CypA介导的MVV Vif-E3泛素形成 连接酶（Aim 2），我们将组装MVV Vif/CypA/E3连接酶复合物，有或没有A3底物，确定 他们的高分辨率结构，并执行生化和功能验证我们的结构 意见。本文还研究了衣壳蛋白对MVV Vif-E3连接酶组装和活化的影响。 追究描述慢病毒Vif-E3泛素募集PPP 2 R5/PP 2A的机制 连接酶（Aim 3），我们将研究PPP 2 R5蛋白对CBFβ介导的 HIV-1 Vif和CypA介导的MVV Vif-E3连接酶，获得高分辨率结构，并进行结构- 引导验证。我们全面的研究设计提供了一个强大的方法，将产生 这是对慢病毒Vif分子的多种功能的前所未有的了解。

PROJECT DESCRIPTION The APOBEC3 (A3) family of proteins are cellular cytidine deaminases that suppress human immunodeficiency virus type 1 (HIV-1) infection by hypermutation of viral reverse transcripts and physically blocking reverse transcription. To evade this host defense mechanism, HIV-1 expresses the virion infectivity factor (Vif), which hijacks a cellular E3 ubiquitin ligase complex and targets A3 proteins (A3F/G/H/D) for proteasome-mediated degradation. Besides degrading A3s, HIV-1 Vif also causes G2 cell cycle arrest by targeting multiple protein phosphatase 2A (PP2A) regulators (PPP2R5 proteins) for degradation. Adding to the complexity of Vif-host protein interactions, HIV-1 Vif utilizes the transcription factor CBFβ as a non-canonical cofactor, while maedi-visna virus (MVV) Vif co-opts the prolyl isomerase cyclophilin A (CypA) instead. Our goal is to establish the biochemical and structural principles for the multifaceted activities of lentiviral Vif molecules that recruit cellular factors to degrade host proteins via ubiquitin-proteasome pathways. To achieve our goal, we will use a combination of biochemical, biophysical, structural biology, and cellular functional techniques. To establish the mechanisms by which A3 proteins are targeted by the HIV-1 Vif (Aim1), we will determine high- resolution structures of the A3-Vif-E3 interaction complexes, validate these structures by structure-guided mutagenesis experiments in vitro and in vivo, and interrogate the molecular determinants of Vif/A3/E3 ligase assembly and activation. In addition, we will also study the degradation-independent mode of Vif inhibition of A3 deamination and antiviral activities. To better understand CypA-mediated formation of MVV Vif-E3 ubiquitin ligase (Aim2), we will assemble MVV Vif/CypA/E3 ligase complexes with or without A3 substrates, determine their high-resolution structures, and perform biochemical and functional validations of our structural observations. The influences of capsid proteins on the assembly and activation of MVV Vif-E3 ligase will also be investigated. To delineate the mechanisms of PPP2R5/PP2A recruitment by lentiviral Vif-E3 ubiquitin ligases (Aim3), we will investigate the effects of PPP2R5 proteins on the assemblies of the CBFβ-mediated HIV-1 Vif and CypA-mediated MVV Vif-E3 ligases, obtain high-resolution structures, and perform structure- guided validations. Our comprehensive research design provides a robust approach that will generate unprecedented insights into the diverse functions of lentiviral Vif molecules.