Project 3

项目3

• 批准号: 10506989
• 负责人: Alexander Marson
• 金额: $ 98.93万
• 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/10506989
• 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 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; 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; base; cell type; combinatorial; complex data; flexibility; genetic approach; genetic evolution; genetic information; genome-wide; high throughput screening; human pathogen; human reference genome; improved; in vivo; innovation; innovative technologies; insertion/deletion mutation; insight; macrophage; mutant; mutation screening; novel; pathogen; protein complex; protein protein interaction; protein structure; repaired; screening; structural biology; 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：艾滋病毒附件和调节复合物 项目3：艾滋病毒限制因素的遗传和演变 总结 宿主病毒界面的良好描述的效应复合物已在结构上表征的基础上，实验室， 适应的HIV毒株和在HIV感染的细胞系模型中发现的单一宿主序列。然而，细胞系 不反映HIV感染在原代CD 4 + T细胞中的功能不同的分子生物学， 巨噬细胞此外，HIV是最多样化的人类病原体之一，但大多数可用的结构性免疫缺陷病毒（HIV）是一种免疫缺陷病毒。 数据是基于少数实验室适应的菌株，而不是原始分离株，以及宿主-病原体蛋白质界面 在进化史上是会改变的在项目3中，我们将使用 从多个供体中分离的原代细胞，用不同的HIV毒株感染它们，然后鉴定新的 来自多种全基因组合并筛选的菌株特异性前病毒和抗病毒宿主因子。合作 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， 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、合并的CRISPRko来识别功能相关的HIV效应子， 在原代CD 4 +T细胞中进行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 通过在原代活化的CD 4 +T细胞中进行多重阵列编辑进行验证， 将通过在静息CD 4 + T细胞中进行阵列编辑来进一步研究相互作用，以检查它们在HIV中的作用。 延迟。确定原代CD 4 + T细胞所需的宿主复合物的功能和结构， 在巨噬细胞中，我们将进行高通量阵列CRISPR-Cas9基因编辑， 因子，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 效应蛋白将通过HARC内源性蛋白结构（HEPS）平台（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 已知宿主-病原体蛋白质界面的突变扫描的结构限制 时间将评估阳性选择下的残基在蛋白质表面和已知复合物上的位置 并通过敲入多克隆修复文库在原代T细胞中进行突变扫描 （Genetics Core）。每种修复结果对病毒复制的影响将用于从遗传学上定义 相互作用的灵活性，并告知宿主-病原体界面的进化轨迹。总的来说， 项目3将确定新的HIV-宿主因子复合物，用于治疗性的结构询问。 有针对性的，并揭示了病毒进化，传播和发病机制的新见解。