Regulation of Vif and Rewiring of Host Pathways

慢病毒只编码十几种蛋白质，但这些蛋白质是如何合作实现持久的 灵长类动物和人类的感染是复杂的，涉及一系列蛋白质相互作用和翻译后 修改。众所周知，病毒蛋白是多功能的，但分离和描述这些 功能及其相应的分子机制仍然是该领域的主要挑战。至 更好地理解HIV VIF的多功能本质，本项目使用了一套系统和公正的 确定VIF参与哪些宿主因素和途径以及这些变化如何的方法 影响病毒复制。VIF最关键的功能是通过中和病毒来促进病毒的传染性 APOBEC3(A3)家族的限制因子，靶向它们被26S蛋白酶体降解。Vif Vif 灵长类慢病毒的蛋白质通过宿主因子劫持转录辅助因子来实现这一点 Cbfβ和库林5环泛素E3连接酶(CRL5)。VIF与CBF、β和CRL5的相互作用 模板其折叠和功能，暴露与A3家族成员特定相互作用的残基，从而 在多泛素化和降解方面。尽管在这一领域进行了十多年的研究，但泛素机器 对A3周转的调控还没有完全描述。此外，几项研究表明，VIF可能会对 通过一种未知机制调节非降解性A3抑制的作用。同时， CD_4~+T细胞全基因组转录组研究显示Vif具有下调CbF的能力 依赖基因，包括A3家族成员，但如何实现这一点的机制也不是 明白了。在这里，我们建议通过三个相关但不同的目标来解决VIF生物学中的这些关键差距：1) 我们将探讨Vif通过泛素化重新连接宿主细胞先天免疫的程度以及这种免疫是如何进行的 回到转录控制；2)我们将确定Vif E3活性的调节如何依赖于 与促进降解性或非降解性泛素修饰的辅助因子相互作用；以及3)我们将 研究通过Vif介导的细胞重组抑制A3的非降解机制。这 Project结合了最先进的方法来询问被泛素化的蛋白质的补充 VIF；其活性所需的辅酶；以及其使用独特的功能分离的非降解功能 FAB工具。我们发现的生物学意义将通过病毒感染性研究来验证 结合CRISPR在原代细胞中对宿主因子的基因编辑。该项目的完成将提供 一个单一的机器，即Vif E3连接酶，如何通过扰动作为纽带的综合观点 通过多种宿主途径逃避免疫系统，促进艾滋病毒的感染性。

Lentiviruses encode just over a dozen proteins, but how these proteins cooperate to achieve a persistent infection in primates and humans is complex, involving a network of protein interactions and post-translational modifications. It is well appreciated that viral proteins are multifunctional, but separating and describing these functions, along with their corresponding molecular mechanisms, is still a major challenge for the field. To better understand the multifunctional nature of HIV Vif, this project uses a set of systematic and unbiased methodologies to determine which host factors and pathways are engaged by Vif and how these changes impact viral replication. The most critical function of Vif is to promote viral infectivity by neutralizing the APOBEC3 (A3) family of restriction factors, targeting them for degradation by the 26S proteasome. The Vif protein of primate lentiviruses achieves this through host factor hijacking of both the transcription cofactor CBFβ as well as the Cullin5-RING ubiquitin E3 ligase (CRL5). Interactions of Vif with CBFβ and CRL5 templates its folding and function, exposing residues for specific interactions with A3 family members resulting in polyubiquitination and degradation. Despite over a decade of research in this area, the ubiquitin machinery regulating A3 turnover has not yet been fully described. Furthermore, several studies indicate Vif may play a role in regulating non-degradative A3 inhibition through an uncharacterized mechanism. At the same time, genome wide transcriptome studies in CD4+ T-cells reveal that Vif has the capacity to downregulate CBF dependent genes, including the A3 family members, but the mechanism of how this is achieved is also not understood. Here, we propose to tackle these critical gaps in Vif biology in three related, yet distinct aims: 1) we will probe the degree to which Vif re-wires host cell innate immunity by ubiquitination and how this feeds back onto transcription control; 2) we will determine how regulation of the Vif E3 activity depends on interactions with co-factors that promote degradative or non-degradative ubiquitin modifications; and 3) we will examine non-degradative mechanisms of A3 inhibition through Vif-mediated cellular reorganization. This project combines state-of-the art methods to interrogate the complement of proteins that are ubiquitinated by Vif; the coenzymes required for its activity; and its non-degradative functions using unique separation-of-function Fab tools. The biological significance of our findings will be validated by viral infectivity studies in conjunction with gene editing of host factors by CRISPR in primary cells. Completion of this project will provide a comprehensive view of how a single machine, namely the Vif E3 ligase, can serve as a nexus by perturbing multiple host pathways to evade the immune system and promote HIV infectivity.