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 配件和调节复合体 项目 3：HIV 限制因素的遗传学和进化 概括 详细描述的效应复合物的宿主-病毒界面已根据实验室特征进行了结构表征。 HIV 适应株和 HIV 感染细胞系模型中发现的单宿主序列。然而，细胞系 不反映原代 CD4+ T 细胞中 HIV 感染的功能独特的分子生物学，并且 巨噬细胞。此外，艾滋病毒是最多样化的人类病原体之一，但大多数可用的结构性病原体 数据基于少数经过实验室改造的菌株，而不是主要分离株，以及宿主-病原体蛋白质界面 已知在进化史上会发生变化。在项目 3 中，我们将通过使用 从多个捐赠者中分离出原代细胞，并用不同的 HIV 毒株感染他们，然后鉴定新的 来自各种全基因组汇集筛选的菌株特异性前病毒和抗病毒宿主因子。合作中 凭借 Genetics Core，我们将应用一套创新技术对人类原代细胞进行基因编辑， 研究正选择下的限制因素并确定对 HIV 重要的相互作用 限制。 我们的项目将使用全基因组、汇集的 CRISPRko 来识别功能相关的 HIV 效应子的宇宙， 原代 CD4+T 细胞中的 CRISPRi 和 CRISPRa 筛选。这些结果将与以前的数据集集成 定义功能性 HIV 宿主网络（使用计算核心）。新的宿主因子候选者将是 通过原代激活 CD4+T 细胞中的多重阵列编辑进行验证，产生高置信度 将通过对静息 CD4+ T 细胞进行阵列编辑来进一步研究相互作用因子，以检查它们在 HIV 中的作用 延迟。定义原代 CD4+ T 细胞所需的宿主复合物的功能和结构， 巨噬细胞，我们将对经过功能验证的高通量阵列 CRISPR-Cas9 基因编辑 因素并用不同的病毒株和突变体感染两种细胞类型。特定蛋白质相互作用网络 将通过HARC内源蛋白结构（HEPS）平台进一步分析效应蛋白（在 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 通过对已知正在改变的宿主-病原体蛋白质界面进行突变扫描来进行结构限制 时间。将评估正选择下的残基在蛋白质表面和已知复合物上的位置 界面并通过多克隆修复文库的敲入对原代 T 细胞进行突变扫描 （遗传学核心）。每个修复结果对病毒复制的影响将用于从基因角度定义 相互作用的灵活性，并告知宿主-病原体界面的进化轨迹。总之， 项目 3 将鉴定新型 HIV 宿主因子复合物，用于结构研究，可用于治疗 有针对性地揭示病毒进化、传播和发病机制的新见解。