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: 10326954
• 负责人: Yong Xiong
• 金额: $ 50.25万
• 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/10326954
• 关键词: 3-Dimensional; Antiviral Agents; 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; Human Parainfluenza Virus 2; Immune; Immune system; In Vitro; Infection; Integration Host Factors; 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 Replication; Visna-maedi virus; Work; X-Ray Crystallography; cellular targeting; cofactor; combat; design; experimental study; factor A; in vivo; inhibitor/antagonist; insight; multicatalytic endopeptidase complex; novel therapeutics; pathogen; protein degradation; reconstruction; recruit; structural biology; three dimensional structure; tool; 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.

项目说明 APOBEC3(A3)家族蛋白是细胞胞苷脱氨酶，能抑制人 免疫缺陷病毒1型(HIV-1)通过病毒逆转录超突变和物理感染 阻止逆转录。为了逃避这种宿主防御机制，HIV-1表达了病毒粒子的传染性 因子(Vif)，劫持细胞E3泛素连接酶复合体，靶向A3蛋白(A3F/G/H/D) 蛋白酶体介导的降解。除了降解A3外，HIV-1 Vif还通过以下方式导致G2细胞周期停滞 靶向多个蛋白磷酸酶2A(PP2A)调节剂(PPP2R5蛋白)进行降解。添加到 Vif与宿主蛋白相互作用的复杂性，HIV-1Vif利用转录因子Cbfβ作为非规范的 辅因子，而Maedi-Visna病毒(MVV)Vif共用Pro异构酶亲环素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 通过泛素-蛋白酶体途径招募细胞因子降解宿主蛋白。为了实现我们的目标， 我们将结合使用生化、生物物理、结构生物学和细胞功能技术。至 建立A3蛋白被HIV-1 Vif(Aim1)靶向的机制，我们将确定高- A3-Vif-E3相互作用络合物的拆分结构，通过结构指导验证这些结构 Vif/A3/E3连接酶的体内外诱变实验及其分子决定因素的研究 组装和激活。此外，我们还将研究Vif抑制的非降解模式。 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)，我们将组装MVV Vif/CypA/E3连接酶复合体，有或没有A3底物，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连接酶组装和激活的影响 被调查。慢病毒Vif-E3泛素对PPP2R5/PP2A募集机制的研究 连接酶(AIM3)，我们将研究PPP2R5蛋白对脑血流量β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，并执行结构- 引导式验证。我们全面的研究设计提供了一种强大的方法，将产生 对慢病毒Vif分子的各种功能的前所未有的见解。