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

慢病毒 Vif 与宿主分子之间的多方面相互作用增强病毒感染力

• 批准号: 10600207
• 负责人: Yong Xiong
• 金额: $ 3.77万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10600207
• 关键词: Biochemical; Biophysics; Capsid Proteins; Cell Cycle Arrest; Complex; Cyclophilin A; Cytidine Deaminase; Deamination; Goals; HIV; HIV-1; Host Defense Mechanism; Immune; In Vitro; Infection; Integration Host Factors; Mediating; Molecular; Mutagenesis; Pathway interactions; Peptidylprolyl Isomerase; Protein Family; Protein phosphatase; Proteins; Research Design; Resolution; Reverse Transcription; Structure; Techniques; Therapeutic; Transcript; Ubiquitin; Validation; Viral; Viral Physiology; Viral Proteins; Virion; Virus Diseases; Virus Inhibitors; Visna-maedi virus; Work; cofactor; design; experimental study; factor A; in vivo; insight; multicatalytic endopeptidase complex; recruit; structural biology; transcription factor; ubiquitin-protein ligase

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.

项目描述 蛋白质的apobec3（a3）家族是抑制人的细胞胞化蛋白 免疫缺陷病毒1型（HIV-1）感染通过病毒逆转录和物理上的超数 阻止逆转录。为了逃避这种宿主防御机制，HIV-1表达了病毒体感染 因子（VIF），劫持细胞E3泛素连接酶复合物并靶向A3蛋白（A3F/g/h/d） 蛋白酶体介导的降解。除了降解A3S外，HIV-1 VIF还通过 靶向多种蛋白质磷酸酶2A（PP2A）调节剂（PPP2R5蛋白）以降解。添加到 HIV-1 VIF使用转录因子CBFβ作为非典型的，HIV-1 VIF的复杂性 辅因子，而Maedi-visna病毒（MVV）VIF选择了丙酸异构酶环磷脂A（CYPA）。我们的目标 是为慢病毒VIF分子的多方面活性建立生化和结构原理 该募集细胞因子通过泛素 - 蛋白酶体途径降解宿主蛋白。为了实现我们的目标， 我们将结合生化，生物物理，结构生物学和细胞功能技术。到 建立A3蛋白是由HIV-1 VIF靶向的机制（AIM1），我们将确定高 - A3-VIF-E3相互作用复合物的分辨率结构，通过结构引导验证这些结构 在体外和体内进行诱变实验，并询问VIF/A3/E3连接酶的分子确定剂 组装和激活。此外，我们还将研究对VIF抑制的降解模式 A3脱氨和抗病毒活动。更好地了解MVV VIF-E3泛素的CYPA介导的形成 连接酶（AIM2），我们将组装有或没有A3底物的MVV VIF/CYPA/E3连接酶配合物，确定 它们的高分辨率结构，并对我们的结构进行生化和功能验证 观察。衣壳蛋白对MVV VIF-E3连接酶的组装和激活的影响也将 被调查。描绘慢病毒VIF-E3泛素的PPP2R5/PP2A的机制 连接酶（AIM3），我们将研究PPP2R5蛋白对CBFβ介导的组件的影响 HIV-1 VIF和CYPA介导的MVV VIF-E3连接酶，获得高分辨率结构，并执行结构 - 指导验证。我们的全面研究设计提供了一种强大的方法，该方法将产生 对慢病毒VIF分子的潜水功能的前所未有的见解。