Project 1

项目1

• 批准号: 10666666
• 负责人: John D Gross
• 金额: $ 90.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10666666
• 关键词: 26S proteasome; APOCEC3G gene; Acquired Immunodeficiency Syndrome; Affect; Architecture; Binding; Binding Proteins; Binding Sites; Biochemical; Biochemistry; Biological Assay; Biology; Birth; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cell Cycle Arrest; Cell Line; Cells; Cercocebus; Chromatin; Complementary DNA; Complex; Cryoelectron Microscopy; Cullin Proteins; Cytidine Deaminase; Data; Deaminase; Dimerization; Down-Regulation; Drug Design; Evolution; Family; Family member; Genetic; Genetic Transcription; HIV; HIV Infections; HIV-1; Hominidae; Human; Integration Host Factors; Intercept; Knock-out; Length; Molecular; Molecular Conformation; Monkeys; Mutagenesis; Phosphoric Monoester Hydrolases; Polyubiquitination; Primates; Process; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; Proteomics; Proviruses; RNA; Resolution; Reverse Transcription; Route; SIV; Sampling; Structure; System; Testing; Ubiquitin; Ubiquitin-mediated Proteolysis Pathway; Ubiquitination; Viral; Viral Proteins; Virion; Virus; Virus Replication; Work; antagonist; arms race; cross-species transmission; dimer; experimental study; genetic approach; innovation; molecular mass; multitask; mutation screening; novel; pandemic disease; prevent; receptor; reconstruction; structural biology; ubiquitin-protein ligase; vif Gene Products

THE HARC CENTER: HIV ACCESSORY AND REGULATORY COMPLEXES PROJECT 1: STRUCTURE AND EVOLUTION OF THE VIF-APOBEC3 COMPLEX SUMMARY In Project 1 we will elucidate novel structural aspects of the APOBEC3 (A3) family of restriction factors and how they are antagonized by the HIV accessory protein Vif. Primate Vif targets A3’s for degradation by the 26S proteasome, but it is unknown how Vif intercepts A3 packaging complexes. It has been suggested that Vif binds different A3 family members through three different interfaces, but whether these binding sites are independent or dependent on one another is unclear. In previous studies, we uncovered that Vif forms functional interactions with additional host factors, including regulatory subunits of PP2A, components of the chromatin-modifying and transcriptional machinery, and regulators of ubiquitin-mediated proteolysis. We will now investigate how Vif neutralizes different A3 family members to promote efficient viral replication, and how adaptations in Vif enabled neutralization of A3G in hominid primates and how these adaptations affect the ability of human A3G to escape HIV-1. We will determine the structure of A3G and PP2A regulatory subunits bound to Vif using cryo-EM (Structural Biology Core), as well as deep mutational scanning (DMS) (Genetics Core), to uncover the mechanisms by which Vif recognizes different substrates and multitasks the degradation of A3 and PP2A subunits. The functional significance of structural observations will be further tested using viral and biochemical assays, and DMS in primary CD4+ T cells will explore tradeoffs between the ability of Vif to neutralize specific A3 family members vs. others. We will also use cryo-EM (Structural Biology Core), functional studies, and DMS (Genetics Core) to determine how Vif’s ability to engage restriction factors is rewired by adaptations allowing cross-species transmission. This is important because A3G and Vif undergo repeated bouts of positive selection and adaptation in what has been termed a ‘molecular arms race’, a process which led to cross-species transmission and the birth of HIV-1. Finally, we will investigate the mechanism of A3 packaging in the absence of Vif by determining composition and architecture of A3 packaging complexes, a long-standing question in the field. We will use the HEPS platform to discover host and viral proteins required for packaging of newly synthesized A3 family members (Proteomics Core). CRISPR-Cas9 and mutagenesis will determine the functional significance of the A3 packaging complex (Genetics Core). Cryo-EM studies will be performed on the packaging complex (Structural Biology Core). These approaches will provide snapshots of A3 family members en route to packaging and define how Vif intercepts these structures to promote viral infectivity. Discoveries made by Project 1 will enable rational drug design to target HIV-1 from establishing replication-competent proviruses by utilizing the restriction potential of A3 family members.

HARC中心：HIV附件和调控复合体 项目1：VIF-APOBEC3复合体的结构和演化 摘要 在项目1中，我们将阐明APOBEC3(A3)限制因子家族的新结构方面以及如何 他们被HIV辅助蛋白Vif所对抗。灵长类Vif目标A3‘S被26S降解 蛋白酶体，但尚不清楚Vif如何拦截A3包装复合体。有人建议，Vif绑定 不同的A3家族成员通过三种不同的接口，但这些结合位点是否独立 或者是相互依赖还不清楚。在以前的研究中，我们发现Vif形成功能相互作用 与其他宿主因子，包括PP2A的调节亚单位，染色质修饰和 转录机制，以及泛素介导的蛋白分解的调节器。我们现在将调查VIF是如何 中和不同的A3家族成员以促进有效的病毒复制，以及如何在VIF中启用适应 A3G在人灵长类动物中的中和以及这些适应如何影响人类A3G逃逸的能力 HIV-1。 我们将使用冷冻-EM(结构分析)来确定与VIF结合的A3G和PP2A调节亚基的结构 生物核心)，以及深度突变扫描(DMS)(遗传学核心)，通过以下方式揭示机制 其中Vif识别不同的底物，并多任务降解A3和PP2A亚基。功能界别 结构观察的意义将进一步通过病毒和生化分析以及DMS在 原代CD4+T细胞将探索Vif中和特定A3家族成员的能力与 其他。我们还将使用冷冻-EM(结构生物学核心)、功能研究和DMS(遗传学核心)来 确定Vif利用限制因子的能力是如何通过允许跨物种的适应而重新连接的 变速箱。这一点很重要，因为A3G和VIF经历了反复的积极选择和适应 在一场被称为“分子军备竞赛”的过程中，这一过程导致了跨物种的传播和 HIV-1的诞生。最后，我们将通过确定在没有VIF的情况下A3包装的机制来研究 A3包装复合体的组成和架构，是该领域长期存在的问题。我们将使用 HEPS平台发现包装新合成的A3家族所需的宿主和病毒蛋白 成员(蛋白质组学核心)。CRISPR-Cas9和突变将决定该基因的功能意义 A3包装复合体(遗传学核心)。将对包装复合体进行低温电磁研究 (结构生物学核心)。这些方法将提供A3家庭成员在前往 包装并定义Vif如何拦截这些结构以促进病毒感染性。由以下人员做出的发现 项目1将使合理的药物设计能够针对HIV-1，通过以下方式建立具有复制能力的前体 利用A3家族成员的限制潜力。