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: 10037560
• 负责人: Paul D. Bieniasz
• 金额: $ 145.19万
• 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/10037560
• 关键词: AIDS/HIV problem; 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; Electron Microscopy; Equipment; Event; Fluorescence Microscopy; Genes; Genetic; Genome engineering; Goals; HIV; HIV Genome; HIV therapy; HIV-1; Human; Image; Individual; Infection; Integrase; Integrase Inhibitors; Integration Host Factors; Kinetics; Knowledge; Label; Life Cycle Stages; Link; Location; Maps; Measurement; Measures; Melissa; Methods; Microscopy; Modeling; Molecular; Mutation; Nature; Nuclear; Nuclear Pore; Nuclear Pore Complex; Paint; Pathway interactions; Phase; Phenotype; Play; Positioning Attribute; Prevention; Process; Provirus Integration; Proviruses; RNA-Directed DNA Polymerase; Research; Research Personnel; Resolution; Reverse Transcription; Role; Route; Series; Specialist; Structure; Systems Biology; Techniques; Technology; Therapeutic; Translating; Treatment Protocols; Viral; Virion; Virus; base; cell immortalization; cell type; cryogenics; drug development; experience; imaging approach; improved; in vivo; innovation; insight; live cell imaging; medical specialties; member; molecular imaging; multidisciplinary; novel; particle; population based; pre-exposure prophylaxis; prevent; small molecule inhibitor; therapeutic target; tool; trafficking; treatment optimization; uptake; virology

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从病毒融合到前病毒整合的步骤 并且代表了关键的治疗靶点。HIV编码的逆转录酶的小分子抑制剂， 整合酶是许多治疗性治疗方案和暴露前预防中心组分。 尽管这些步骤的治疗重要性，该领域仍然缺乏共识，在几个突出的 问题包括贩运和脱壳如何与逆转录有关， 前病毒通过核孔，什么样的宿主因子参与了这些过程， 区分成功感染和失败感染的病毒。由于效率低下， 和相对随机的早期阶段复制的性质，只有一小部分（~15%）的颗粒进入 融合后的细胞质将导致成功的前病毒整合。因此，基于群体的测定， 衡量大多数病毒的行为可能会也可能不会真正捕捉到成功病毒的行为。然而，尽管如此， 技术限制历来要求依赖于基于群体测定、永生化细胞系 模型和间接测量的生物过程，其基本假设不一定 反映了生物学上的先验。直到最近才在单粒子追踪、分子成像、基因 编辑和结构确定允许研究人员克服这些限制，但这些专业化的 迄今为止，科学技术还没有被整合到一起来回答艾滋病毒生物学中的这些关键问题。这里我们 组建一个艾滋病毒研究人员团队，他们在这些强大的方法方面具有互补的专业知识， 定义导致生产性感染的融合和整合之间的相互作用、动力学和动力学。 我们建议利用一种新优化的分子标记方法工具箱， 称为感染性病毒粒子跟踪（IVT），以成像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体与非生产性地进入细胞的病毒体分离。 其他专业技术包括原代细胞CRISPR-Cas9基因编辑和低温电子 将利用显微镜来询问沿着各个组件的结构和功能 转化为生产性感染运用这一系列新颖和创新的工具，方法和设备，我们的目标是 目的：1）在优化的细胞培养模型和原代细胞培养模型中定义HIV从融合到整合的感染途径 人靶细胞; 2）确定宿主粘附因子和病毒组分在人靶细胞的过程中的作用。 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，因为它通过逆转录，通过细胞质运输， 进入细胞核，并最终整合到宿主染色体DNA中。作为一个合作团队， 补充专业，解决该领域的关键限制，我们处于独特的地位，使 对我们目前对艾滋病毒复制早期阶段的理解做出了重大贡献。