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：HIV配件和监管综合体项目1：VIF-APOBEC3复合物的结构和演变概括在项目1中，我们将阐明APOBEC3（A3）限制因素家族的新结构方面以及如何它们被HIV辅助蛋白VIF拮抗。灵长类动物VIF靶向26年代的A3降解蛋白酶体，但尚不清楚VIF如何拦截A3包装复合物。有人建议VIF结合通过三个不同的接口不同的A3家族成员，但是这些绑定位点是否独立或彼此依赖不清楚。在先前的研究中，我们发现VIF形成了功能相互作用具有其他宿主因素，包括PP2A的调节亚基，染色质修饰的组成部分和转录机械和泛素介导的蛋白水解的调节剂。我们现在将调查VIF 中和不同的A3家庭成员以促进有效的病毒复制，以及如何启用VIF的适应人类灵长类动物中A3G的中和以及这些适应如何影响人类A3G逃脱的能力 HIV-1。我们将确定使用Cryo-EM（结构性的）A3G和PP2A调节亚基的结构生物学核心）以及深突变扫描（DMS）（遗传学核心），以发现通过 VIF识别不同的底物和多任务降解A3和PP2A亚基的降解。功能结构观测的重要性将通过病毒和生化测定进一步测试，而DMS则将在主要的CD4+ T细胞将探索VIF中和特定A3家族成员VS的能力之间的权衡。其他的。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）通过适应允许跨物种的适应来确定VIF如何参与限制因素的能力传播。这很重要，因为A3G和VIF经历了重复的积极选择和适应在被称为“分子武器竞赛”的过程中，这一过程导致了跨物种的传播和 HIV-1的出生。最后，我们将在没有VIF的情况下研究A3包装的机制 A3包装综合体的组成和结构，这是该领域的长期问题。我们将使用发现新合成的A3家族所需的宿主和病毒蛋白的HEP平台成员（蛋白质组学核心）。 CRISPR-CAS9和诱变将确定 A3包装复合物（遗传学核心）。 Cryo-EM研究将在包装复合物上进行（结构生物学核心）。这些方法将为A3家庭成员提供快照包装并定义VIF如何拦截这些结构以促进病毒感染。发现项目1将使理性的药物设计能够以建立复制能力的预科病为目标的hiv-1 利用A3家庭成员的限制潜力。