Functionally Defining HIV-Host Interactions During the Early HIV-1 Lifecycle

在 HIV-1 生命周期早期从功能上定义 HIV 与宿主的相互作用

• 批准号: 10594493
• 负责人: Paul D. Bieniasz
• 金额: $ 135.17万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10594493
• 关键词: Address; Anti-Retroviral Agents; Area; Behavior; Biochemical; Biological; Biological Assay; Biological Phenomena; Biological Process; Biology; CRISPR/Cas technology; Cell Compartmentation; Cell Culture Techniques; Cell Line; Cell Nucleus; Cell model; Cells; Cellular biology; Complex; Conflict (Psychology); Consensus; Cryoelectron Microscopy; Cytoplasm; DNA; Data; Development; Disparity; Electron Microscopy; Equipment; Event; Fluorescence Microscopy; Genes; Genetic; Genome engineering; Goals; HIV; HIV Genome; HIV therapy; HIV-1; HIV/AIDS; Human; Image; Individual; Infection; Integrase; Integrase Inhibitors; Integration Host Factors; Kinetics; Knowledge; Label; Life Cycle Stages; Link; Location; Maps; Measurement; Measures; Methods; Microscopy; Modeling; Molecular; Mutation; Nature; Nuclear; Nuclear Pore; Nuclear Pore Complex; Paint; Pathway interactions; Persons; Phase; Phenotype; Play; Positioning Attribute; Prevention; Process; Productivity; Provirus Integration; Proviruses; RNA-Directed DNA Polymerase; Research; Research Personnel; Reverse Transcription; Role; Route; Series; Specialist; Structure; Systems Biology; Techniques; Technology; Therapeutic; Translating; Treatment Protocols; Viral; Virion; Virus; Visualization; cell immortalization; cell type; drug development; experience; imaging approach; improved; in vivo; innovation; insight; live cell imaging; medical specialties; member; molecular imaging; multidisciplinary; novel; particle; permissiveness; population based; pre-exposure prophylaxis; prevent; small molecule inhibitor; therapeutic target; tool; trafficking; treatment optimization; ultra high resolution; uptake

ABSTRACT: The early phase of the HIV lifecycle encompasses the steps from virus fusion to provirus integration and represents a critical therapeutic target. Small molecule inhibitors of HIV encoded reverse transcriptase and integrase are central components of many therapeutic treatment regimens and pre-exposure prophylaxis. Despite the therapeutic importance of these steps, the field still lacks consensus on several outstanding questions including how trafficking and uncoating are linked to reverse transcription, how and in what state the provirus transits through the nuclear pore, what host factors are involved in these processes, and what distinguishes between a virus that will establish successful infection and one that will fail. Due to the inefficient and relatively stochastic nature of early phase replication, only a small percentage (~15%) of particles that enter the cytoplasm after fusion will result in successful provirus integration. As a result, population-based assays that measure what most viruses do may or may not actually capture what successful viruses do. Nevertheless, technical limitations have historically mandated a reliance on population-based assays, immortalized cell line models, and indirect measurements of biological processes whose underlying assumptions don’t necessarily reflect the biological priors. Only recently have innovations in single-particle tracking, molecular imaging, gene editing, and structural determination allowed for researchers to overcome these limitations, but these specialized technologies have not yet been brought together to answer these critical questions in HIV biology. Here, we assemble a team of HIV researchers with complementary expertise in these powerful approaches to dissect and define the interactions, kinetics, and dynamics between fusion and integration that result in productive infection. We propose to leverage a newly optimized toolbox of molecular labeling methods, a technique collectively termed Infectious Virion Tracking (IVT), to image and track the behavior of individual viral components, ultimately separating individual virions that result in successful infection from those that enter the cell non-productively. Additional specialized technologies including primary cell CRISPR-Cas9 gene editing and cryogenic electron microscopy will be leveraged to interrogate the structure and function of individual components along the route to productive infection. Wielding this novel and innovative series of tools, approaches, and equipment, we aim to: 1) Define the infectious pathway of HIV from fusion to integration in optimized cell culture models and primary human target cells; 2) Determine the role of host permissivity factors and viral components in the processes of the early phase of the HIV life-cycle; and 3) Visualize and define the structure of the viral based machines associated with the HIV genome as it progresses through reverse transcription, traffics through the cytoplasm, enters the nucleus, and ultimately integrates in the host chromosomal DNA. As a collaborative team with complementary specialties that address critical limitations in the field, we are in a unique position to make significant contributions to our current understanding of the early phases of HIV replication.

摘要：HIV生命周期的早期阶段包括病毒融合到病毒整合的步骤 并代表关键的治疗靶点。 HIV编码的逆转录酶的小分子抑制剂和 整合酶是许多治疗治疗方案和暴露前预防的中心成分。 尽管这些步骤具有治疗性的重要性，但该领域仍然在几个出色的 问题在内，包括贩运和脱衣的方式与逆转录有关，如何以及处于哪种状态 病毒通过核孔过渡，这些过程中涉及哪些宿主因素以及什么 区分将建立成功感染的病毒和将失败的病毒。由于效率低下 和早期复制的相对随机性质，只有一小部分（〜15％）进入 融合后的细胞质将导致成功的病毒整合。结果，基于人群的测定法 测量大多数病毒的工作可能或可能不会实际捕获成功病毒的工作。尽管如此， 历史上，技术限制已授予对基于人群的测定，永生的细胞系的缓解 模型和间接测量生物过程的基础假设不一定是 反映生物学先验。直到最近才进行单粒子跟踪，分子成像，基因的创新 编辑和结构性确定允许研究人员克服这些局限性，但是这些专业 尚未将技术汇集在一起​​来回答艾滋病毒生物学中的这些关键问题。在这里，我们 在这些有力的剖析和剖析和 定义导致产品感染的融合与整合之间的相互作用，动力学和动力学。 我们建议利用分子标记方法的新优化的工具箱，这是一种统称的技术 称为感染性病毒体跟踪（IVT），以形象和跟踪单个病毒成分的行为，最终 将导致成功感染的单个病毒与非生产性进入细胞的病毒分离。 其他专业技术，包括原代细胞CRISPR-CAS9基因编辑和低温电子 显微镜将被利用以询问沿路线的单个组件的结构和功能 生产感染。我们掌握了这一新颖而创新的工具，方法和设备，我们的目标 TO：1）定义从融合到优化细胞培养模型中融合到整合的HIV的传染途径 人类靶细胞； 2）确定宿主许可因素和病毒成分在过程中的作用 艾滋病毒生命周期的早期阶段； 3）可视化和定义基于病毒的机器的结构 与HIV基因组相关，它通过逆转录，经过细胞质的运输而发展， 输入细胞核，并最终整合到宿主染色体DNA中。作为与 解决该领域关键限制的互补专业，我们处于独特的位置 我们对当前对HIV复制早期阶段的理解做出了重大贡献。