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

基本信息

批准号：
10640135
负责人：
Yong Xiong
金额：
$ 47.83万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10640135
关键词：
3-Dimensional Binding Biochemical Biophysics CD4 Positive T Lymphocytes Capsid Proteins Cell Cycle Arrest Classification Communities Comparative Study Complex Cryoelectron Microscopy Cyclophilin A Cytidine Deaminase Deamination Degradation Pathway Evolution Fluorescence Goals HIV HIV-1 Host Defense Mechanism Immune Immune system In Vitro Infection Integration Host Factors Invaded Knowledge Mammals Mediating Methods Molecular Mutagenesis PPP2R5A gene Pathway interactions Peptidylprolyl Isomerase Primate Lentiviruses Protein Family Protein Isoforms Protein phosphatase Proteins Research Research Design Research Project Grants Resolution Reverse Transcription Sheep Spectrum Analysis Structure System Techniques Therapeutic Transcript Ubiquitin Validation Viral Viral Physiology Viral Proteins Virion Virus Diseases Virus Inhibitors Virus Replication Visna-maedi virus Work X-Ray Crystallography cellular targeting cofactor combat design experimental study factor A in vivo inhibitor insight multicatalytic endopeptidase complex novel therapeutics pathogen reconstruction recruit structural biology tool transcription factor ubiquitin-protein ligase

项目摘要

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 APOBEC3 (A3) 蛋白家族是细胞胞苷脱氨酶，可抑制人类 1 型免疫缺陷病毒 (HIV-1) 通过病毒逆转录物超突变和物理感染为了逃避这种宿主防御机制，HIV-1 表达病毒粒子的感染性。因子 (Vif)，它劫持细胞 E3 泛素连接酶复合物并靶向 A3 蛋白 (A3F/G/H/D) 除了降解 A3 外，HIV-1 Vif 还通过蛋白酶体介导的降解导致 G2 细胞周期停滞。靶向多种蛋白磷酸酶 2A (PP2A) 调节剂（PPP2R5 蛋白）进行降解。鉴于 Vif-宿主蛋白相互作用的复杂性，HIV-1 Vif 利用转录因子 CBFβ 作为非规范因子辅因子，而梅迪-维斯纳病毒 (MVV) Vif 则选择脯氨酰异构酶亲环蛋白 A (CypA) 作为我们的目标。旨在建立慢病毒Vif分子多方面活性的生化和结构原理招募细胞因子通过泛素蛋白酶体途径降解宿主蛋白以实现我们的目标，我们将结合使用生物化学、生物物理、结构生物学和细胞功能技术。建立 HIV-1 Vif (Aim1) 靶向 A3 蛋白的机制，我们将确定高 A3-Vif-E3 相互作用复合物的解析结构，通过结构引导验证这些结构体外和体内诱变实验，并探讨 Vif/A3/E3 连接酶的分子决定因素此外，我们还将研究 Vif 抑制的降解独立模式。 A3 脱氨基和抗病毒活性为了更好地了解 CypA 介导的 MVV Vif-E3 泛素的形成。连接酶 (Aim2)，我们将在有或没有 A3 底物的情况下组装 MVV Vif/CypA/E3 连接酶复合物，确定他们的高分辨率结构，并对我们的结构进行生化和功能验证衣壳蛋白对 MVV Vif-E3 连接酶的组装和激活的影响也将受到影响。研究慢病毒 Vif-E3 泛素招募 PPP2R5/PP2A 的机制。连接酶 (Aim3)，我们将研究 PPP2R5 蛋白对 CBFβ 介导的组装的影响 HIV-1 Vif和CypA介导的MVV Vif-E3连接酶，获得高分辨率结构，并进行结构- 我们全面的研究设计提供了一种稳健的方法，可以产生对慢病毒 Vif 分子多种功能的前所未有的见解。