Evolution of the Vif E3 Ubiquitin Ligase

Vif E3 泛素连接酶的演变

基本信息

批准号：
9233738
负责人：
Jennifer Binning
金额：
$ 5.92万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-01 至 2019-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9233738
关键词：
AIDS prevention APOCEC3G gene Address Affect Animals Antiviral Agents Binding Biochemical Biochemistry Biological Biological Assay Case Study Cells Cercopithecidae Chronic Complex Cullin Proteins Data Development Enzymatic Biochemistry Enzymes Event Evolution Fluorescence Polarization HIV HIV-1 Hominidae Human Immune Immune Evasion Immune response Immunity In Vitro Individual Interferometry Link Literature Measures Mediating Modeling Molecular Monkeys Mutation Analysis Negative Staining Pan Genus Pathogenesis Phenotype Polyubiquitination Population Primates Proteins Race Reaction Recording of previous events Research Resolution Role SIV Species Specificity Specificity Structural Models Structure Subfamily lentivirinae Techniques Testing Therapeutic Thermodynamics Ubiquitination Viral Viral Genome Viral Pathogenesis Viral Proteins Virion Virus Diseases X-Ray Crystallography Zoonoses arm base design experimental study factor C global health immunoregulation in vivo innovation insight novel pathogen pressure protein degradation public health relevance stoichiometry structural biology transmission process ubiquitin ligase ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): Evolutionary pressures placed upon lentiviruses, such as HIV, and their affected hosts have resulted in a molecular "arms race" which has shaped both host immunity and pathogen immune evasion strategies. Central to this race is the HIV protein Virion Infectivity Factor (Vif), an immunomodulatory protein critical to the replicatio cycle of nearly all lentiviruses. The primary function of Vif is to counteract the antiviral effect of the host APOBEC3 (A3) innate immune proteins. A3 proteins are restriction factors that inhibit lentiviral replication by inducing hypermutation of the viral genome. Vif antagonizes A3 by hijacking a cellular Cullin-RING ubiquitin ligase, resulting in the ubiquitination and subsequent targeting of APOBEC3 for proteasomal degradation. Despite the central role of Vif in the HIV replication cycle and an extensive body of literature describing HIV pathogenesis, we still do not have a clear molecular understanding of how Vifs bind and ubiquitinate A3. This research plan seeks to combine biochemistry, enzymology, and structural biology to understand how Vif inhibits A3 at a molecular level, and determine how A3 inhibition relates to the evolutionary events that allowed HIV to "jump" from monkeys to humans. Lentiviral Vifs have evolved to specifically counteract the effects of the A3 from their animal hosts. To determine the biochemical basis for species-specific A3 recognition I will use a panel of HIV and SIV Vif E3 ligases and their respective A3G proteins to carry out binding and in vitro ubiquitination assays and address the question: is A3G species-specificity conferred during the ground state, binding to Vif, or the transition state, the catalytic step, of the enzyme catalyzed ubiquitination reactio? To provide the molecular determinants that govern Vifs' ability to confer species-specific recognition of A3G, I will utilize low- and high-resolution structural techniques that allow me to visualize the A3G-Vif interface at atomic resolution. Mutational analyses, using in vitro ubiquitination assays and cell-base infectivity assays, will be used to link structure to phenotype and validate the biological relevance of the structural model. The successful completion of these studies will provide "snapshots" of Vif from different stages of lentivirial evolution, and our combined structural and functional approach will provide detailed insight into the Vif- A3 interaction. Together these studies will allow us to "see" the Vif-A3 interaction, characterize the evolutionary steps required for species-species transmission into human populations, and ultimately aid in the development of innovative therapeutics to counter viral infection.

描述（由申请人提供）：慢病毒（例如 HIV）及其受影响的宿主所承受的进化压力导致了一场分子“军备竞赛”，这场竞赛塑造了宿主免疫和病原体免疫逃避策略，这场竞赛的核心是 HIV 蛋白。病毒粒子感染因子 (Vif)，一种对几乎所有慢病毒复制周期至关重要的免疫调节蛋白。Vif 的主要功能是抵消宿主 APOBEC3 的抗病毒作用。 (A3) 先天免疫蛋白 A3 蛋白是通过诱导病毒基因组超突变来抑制慢病毒复制的限制因子，它通过劫持细胞 Cullin-RING 泛素连接酶来拮抗 A3，导致 APOBEC3 泛素化并随后靶向 APOBEC3 进行蛋白酶体降解。尽管 Vif 在 HIV 复制周期中发挥着核心作用，并且有大量文献描述 HIV 发病机制，但我们仍然对 Vif 没有明确的分子认识。 Vifs 如何结合并泛素化 A3 该研究计划旨在结合生物化学、酶学和结构生物学，以了解 Vif 如何在分子水平上抑制 A3，并确定 A3 抑制如何与允许 HIV 从猴子身上“跳跃”的进化事件相关。慢病毒 Vif 已进化为专门抵消动物宿主 A3 的影响，为了确定物种特异性 A3 识别的生化基础，我将使用一组 HIV 和SIV Vif E3 连接酶及其各自的 A3G 蛋白进行结合和体外泛素化测定，并解决以下问题：A3G 物种特异性是否在酶的基态、与 Vif 结合或过渡态（催化步骤）期间赋予为了提供控制 Vifs 赋予 A3G 物种特异性识别能力的分子决定因素，我将利用低分辨率和高分辨率结构使我能够以原子分辨率可视化 A3G-Vif 界面的技术，使用体外泛素化测定和基于细胞的感染性测定，将用于将结构与表型联系起来。并验证结构模型的生物学相关性，这些研究的成功完成将提供慢病毒进化不同阶段的 Vif“快照”，而我们的结构和功能相结合的方法将提供对 Vif-A3 相互作用的详细了解。研究将使我们能够“看到”Vif-A3 相互作用，表征物种之间向人类传播所需的进化步骤，并最终有助于开发对抗病毒感染的创新疗法。