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。