Project 3

项目3

• 批准号: 10666677
• 负责人: Alexander Marson
• 金额: $ 98.3万
• 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/10666677
• 关键词: Automobile Driving; Biology of HIV Infection; CD4 Positive T Lymphocytes; CRISPR interference; CRISPR library; CRISPR screen; CRISPR-mediated transcriptional activation; CRISPR/Cas technology; Cell Line; Cell Separation; Cell model; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complex; Cryoelectron Microscopy; Data; Data Set; Dependence; Evolution; Genes; Genetic; Genetic Screening; Goals; HIV; HIV Infections; HIV-1; Human; Immune system; Integration Host Factors; Knock-in; Knock-out; Libraries; Life Cycle Stages; Macrophage; Maps; Mass Spectrum Analysis; Membrane Proteins; Methods; Modeling; Molecular; Molecular Biology; Mutation; Nucleic Acids; Outcome; Pathogenesis; Phenotype; Point Mutation; Population; Positioning Attribute; Primates; Proteins; Proteomics; Publishing; Recording of previous events; Reporting; Research; Rest; Roentgen Rays; Role; Structure; System; T-Lymphocyte; Technology; Time; Translating; Translations; Viral; Viral Proteins; Virus; Virus Replication; arms race; cell type; combinatorial; complex data; flexibility; genetic approach; genetic information; genome-wide; high throughput screening; human pathogen; human reference genome; improved; in vivo; innovation; innovative technologies; insertion/deletion mutation; insight; mutant; mutation screening; novel; pathogen; protein complex; protein protein interaction; protein structure; repaired; screening; structural biology; structural determinants; therapeutic target; transmission process

THE HARC CENTER: HIV ACCESSORY AND REGULATORY COMPLEXES PROJECT 3: GENETICS AND EVOLUTION OF HIV RESTRICTION FACTORS SUMMARY Host-virus interfaces of well-described effector complexes have been structurally characterized based on lab- adapted strains of HIV and single host sequences found in cell line models of HIV infection. However, cell lines do not reflect the functionally distinct molecular biology of HIV infections in primary CD4+ T cells and macrophages. Moreover, HIV is one of the most diverse human pathogens, yet most of the available structural data are based on a handful of lab-adapted strains and not primary isolates, and host-pathogen protein interfaces are known to change in evolutionary history. In Project 3, we will investigate host and virus diversity by using primary cells isolated from multiple donors and infecting them with diverse HIV strains, and then identifying novel strain-specific proviral and antiviral host factors from a variety of genome-wide pooled screens. In collaboration with the Genetics Core, we will apply a suite of innovative technologies in gene editing of human primary cells, to investigate restriction factors that are under positive selection and identify interactions important for HIV restriction. Our project will identify the universe of functionally relevant HIV effectors using genome-wide, pooled CRISPRko, CRISPRi and CRISPRa screening in primary CD4+T cells. These results will be integrated with previous datasets to define functional HIV-host networks (with the Computational Core). Novel host factor candidates will be validated by multiplexed, arrayed editing in primary activated CD4+T cells, generating high-confidence interactors that will be further investigated by arrayed editing in resting CD4+ T cells to examine their role in HIV latency. To define the function and structure of host complexes required in primary CD4+ T cells and macrophages, we will perform high-throughput arrayed CRISPR-Cas9 gene editing of functionally validated factors and infect both cell types with different viral strains and mutants. Protein interaction networks of specific effector proteins will be further analyzed through the HARC endogenous protein structure (HEPS) platform (in collaboration with the Proteomics and Structural Biology Cores). Furthermore, we will map evolutionary and structural constraints by mutational scanning of host-pathogen protein interfaces known to be changing over time. Residues under positive selection will be assessed for position on protein surfaces and known complex interfaces and subjected to mutational scanning in primary T cells by knock-in of polyclonal repair libraries (Genetics Core). The impact of each repair outcome on viral replication will be used to genetically define the flexibility of the interaction, and to inform the evolutionary trajectory of host-pathogen interfaces. In summary, Project 3 will identify novel HIV-host factor complexes for structural interrogation that can be therapeutically targeted and reveal new insights in viral evolution, transmission, and pathogenesis.

HARC中心：HIV附件和调控复合体 项目3：艾滋病毒限制因素的遗传学和进化 摘要 描述良好的效应复合体的宿主-病毒界面已经在实验室的基础上进行了结构表征- 在艾滋病毒感染的细胞系模型中发现的艾滋病毒适应株和单一宿主序列。然而，细胞系 不能反映原代CD4+T细胞中HIV感染的不同功能分子生物学 巨噬细胞。此外，艾滋病毒是人类最多样化的病原体之一，但现有的大多数结构性 数据是基于几个实验室适应的菌株，而不是初级分离物，以及宿主-病原体蛋白界面 在进化史上会发生变化。在项目3中，我们将使用以下方法调查宿主和病毒的多样性 从多个捐赠者身上分离出原代细胞，并用不同的HIV毒株感染它们，然后鉴定新的 从各种基因组范围的联合筛选中筛选出特定毒株的前病毒和抗病毒宿主因子。在协作中 有了Genetics Core，我们将在人类原代细胞的基因编辑中应用一套创新技术， 调查积极选择的限制因素并确定对艾滋病毒重要的相互作用 限制。 我们的项目将使用全基因组的、汇集的CRISPRko来识别功能相关的HIV效应器， 原代CD4+T细胞CRISPRi和CRISPRa的筛选这些结果将与先前的数据集进行集成 定义起作用的艾滋病毒宿主网络(具有计算核心)。新的主因子候选者将是 通过对原始激活的CD4+T细胞进行多路复用、阵列编辑验证，生成高置信度 将通过阵列编辑进一步研究静息CD4+T细胞的相互作用，以检查它们在HIV中的作用 延迟。为了确定原代CD4+T细胞所需宿主复合体的功能和结构，以及 巨噬细胞，我们将进行高通量阵列CRISPR-Cas9基因编辑的功能验证 用不同的病毒株和突变体影响和感染这两种细胞类型。特定的蛋白质相互作用网络 效应蛋白将通过HARC内源蛋白结构(HEPS)平台(IN)进一步分析 与蛋白质组学和结构生物学核心的合作)。此外，我们将绘制进化和 已知正在转换的宿主-病原体蛋白界面突变扫描的结构限制 时间到了。阳性选择下的残基将被评估其在蛋白质表面和已知复合体上的位置。 通过敲入多克隆修复文库对原代T细胞进行突变扫描 (遗传学核心)。每一次修复结果对病毒复制的影响将被用来从基因上定义 相互作用的灵活性，并告知宿主-病原体界面的进化轨迹。总而言之， 项目3将确定新的HIV-宿主因子复合体，用于结构讯问，可用于治疗 具有针对性，并揭示病毒进化、传播和发病机制的新见解。