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: 10359682
• 负责人: Paul D. Bieniasz
• 金额: $ 135.51万
• 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/10359682
• 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; Persons; 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

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原病毒整合的步骤