Evolution of the Vif E3 Ubiquitin Ligase

 描述(由申请人提供)：对慢病毒，如艾滋病毒，及其受影响的宿主施加的进化压力已导致分子“军备竞赛”，这形成了宿主免疫和病原体免疫逃避策略。这场竞赛的核心是HIV蛋白病毒感染因子(Vif)，这是一种对几乎所有慢病毒的复制周期至关重要的免疫调节蛋白。Vif的主要功能是对抗宿主APOBEC3(A3)天然免疫蛋白的抗病毒作用。A3蛋白是通过诱导病毒基因组的超突变来抑制慢病毒复制的限制因子。VIF通过劫持细胞库林环泛素连接酶来拮抗A3，导致泛素化和随后靶向APOBEC3的蛋白酶体降解。尽管Vif在HIV复制周期中起着核心作用，并且有大量文献描述了HIV的发病机制，但我们仍然没有清楚的分子理解Vif如何结合和泛化A3。这项研究计划试图将生物化学、酶学和结构生物学结合起来，在分子水平上了解Vif如何抑制A3，并确定A3抑制与允许艾滋病毒从猴子“跳”到人类的进化事件有何关系。慢病毒vif已经进化成特别地中和来自动物宿主的A3的影响。为了确定物种特异性A3识别的生物化学基础，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分析，并解决以下问题：A3G是在基态与Vif结合时授予的，还是在酶催化泛素化反应的过渡期(催化步骤)赋予的物种特异性？为了提供控制VIF识别A3G的能力的分子决定因素，我将利用低分辨率和高分辨率的结构技术，使我能够在原子分辨率下可视化A3G-VIF界面。突变分析，使用体外泛素化分析和基于细胞的感染性分析，将用于将结构与表型联系起来。 并验证了结构模型的生物学相关性。这些研究的成功完成将提供Vif在晶状体进化不同阶段的“快照”，我们的结构和功能相结合的方法将为Vif-A3的相互作用提供详细的洞察。这些研究将使我们能够“看到”Vif-A3相互作用，表征 物种间传播到人类群体所需的进化步骤，并最终有助于开发抗击病毒感染的创新疗法。

 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.