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Project 1

项目1

基本信息

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
项目类别：
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

项目摘要

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：艾滋病毒附件和调节复合物项目1：VIF-APOBEC 3复合物的结构和演变总结在项目1中，我们将阐明APOBEC 3（A3）限制因子家族的新结构方面，以及如何它们被HIV辅助蛋白Vif拮抗。灵长类Vif以A3为目标，通过26 S降解蛋白酶体，但不知道Vif如何拦截A3包装复合物。有人认为，Vif 不同的A3家族成员通过三个不同的界面，但这些结合位点是否独立还是互相依赖，还不清楚。在以前的研究中，我们发现Vif形成功能性相互作用，与其他宿主因子，包括PP 2A的调节亚基，染色质修饰的组分，转录机制和泛素介导的蛋白质水解调节因子。我们现在将研究一下中和不同的A3家族成员，以促进有效的病毒复制，以及Vif中的适应如何使原始灵长类动物A3 G的中和以及这些适应如何影响人类A3 G逃逸的能力 HIV-1 我们将使用cryo-EM（结构分析）确定与Vif结合的A3 G和PP 2A调节亚基的结构。生物学核心），以及深度突变扫描（DMS）（遗传学核心），以揭示机制， Vif识别不同的底物并多任务降解A3和PP 2A亚基。功能结构观察的意义将进一步测试使用病毒和生物化学测定，和DMS在原代CD 4 + T细胞将探索Vif中和特定A3家族成员的能力与他人我们还将使用cryo-EM（结构生物学核心），功能研究和DMS（遗传学核心），确定Vif参与限制因素的能力是如何通过允许跨物种的适应来重新连接的传输这一点很重要，因为A3 G和Vif经历了反复的积极选择和适应在所谓的“分子军备竞赛”中，这一过程导致了跨物种传播和 HIV-1的诞生最后，我们将研究A3包装的机制，在没有Vif的情况下，通过确定 A3包装复合物的组成和结构，这是该领域的一个长期问题。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 Proteomics Core（蛋白质组学核心）CRISPR-Cas9和诱变将决定CRISPR-Cas9的功能意义。 A3包装复合物（遗传学核心）。将对包装复合物进行冷冻EM研究（Structural Biology Core）这些方法将提供A3家族成员的快照，包装并定义Vif如何拦截这些结构以促进病毒感染性。发现者项目1将使合理的药物设计能够通过建立具有复制能力的前病毒来靶向HIV-1，利用A3家族成员的限制潜力。