Imaging of Single HIV-1 Uncoating and Transport to the nucleus

单个 HIV-1 脱壳和转运至细胞核的成像

基本信息

批准号：
9354023
负责人：
Gregory B Melikian
金额：
$ 59.65万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-03-24 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9354023
关键词：
Affect Affinity Agreement Avidity Binding Biochemical Biological Assay Capsid Capsid Proteins Cell Line Cell Nucleus Cell fusion Cells Complex Cone Core Protein Cryoelectron Microscopy Cyclophilin A Cytoplasm DNA Integration DNA biosynthesis DsRed Enzymes Event Genetic Genome Goals HIV-1 Hela Cells Heterogeneity Human Image Imagery Imaging Device Individual Infection Infection prevention Integrase Integration Host Factors Kinesin Kinetics Knock-out Label Length Life Link Mediating Membrane Minor Mutation Nature Nuclear Import Nuclear Pore Nuclear Pore Complex Nucleocapsid Phenotype Process Proteins Publishing RNA-Directed DNA Polymerase Regulation Reverse Transcription Reverse Transcription Inhibition Ribonuclease H Series Stem cells Structure Testing Tube Viral Viral Genome Virion Virus Virus Integration anterograde transport base design flexibility genomic RNA improved insight macrophage member mutant novel novel strategies particle premature prevent spatiotemporal viral DNA

项目摘要

Disassembly of the cone-shaped HIV-1 capsid after virus-cell fusion is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. We have recently developed a novel strategy to visualize HIV-1 uncoating that is based on a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed). CypA-DsRed, which is specifically packaged into virions through the high-avidity binding to the HIV-1 capsid, does not compromise the infectivity. This probe remains associated with cores after virus-cell fusion and is released upon uncoating. Supporting this notion is our finding that the rate of CypA-DsRed loss from individual post-fusion cores is modulated by mutations affecting the core stability and is accelerated by reverse transcription. The CypA-DsRed based imaging assay revealed a biphasic kinetic of HIV-1 uncoating, with a large number of cores shedding the capsid protein shortly after fusion. This assay also revealed marked differences in the uncoating phenotype in HeLa-derived cells and primary human macrophages. We propose to: (1) delineate CypA-DsRed interactions with HIV-1 core through functional and structural studies of CypA-DsRed/capsid complexes; (2) elucidate the relationship between reverse transcription and single core uncoating; and (3) investigate regulation of HIV-1 uncoating by host factors, such as Nup358 and CPSF6. These studies, employing a combination of genetic, biochemical and advanced imaging tools, are expected to provide critical insights into the dynamics and regulation of HIV-1 uncoating in living cells.

病毒融合后的锥形HIV-1帽胶的拆卸是前提条件建立终身感染。 HIV-1输入中的这一步骤，称为无涂层，至关重要理解不佳。我们最近制定了一种新颖的策略，以可视化HIV-1脱落基于衣壳结合宿主蛋白环蛋白A的荧光标记的寡聚形式（CYPA-DSRED）。 CYPA-DSRED，该dsred专门通过高野外包装到病毒体中与HIV-1衣壳的结合不会损害感染力。该探针仍然相关在病毒 - 细胞融合后使用核心，并在脱涂后释放。支持这个概念是我们的发现单个融合后核的CYPA-DSRED损失率由影响核心稳定性的突变，并通过逆转录加速。 CYPA-DSRED 基于成像测定显示HIV-1脱涂的双相动力学，大量核心融合后不久将衣壳蛋白脱落。该测定也揭示了明显的差异 HELA来源的细胞和原代人巨噬细胞中的脱膜表型。我们建议至：（1）通过功能和结构来描述与HIV-1核心的CYPA-DSRED相互作用 CYPA-DSRED/CAPSID复合物的研究；（2）阐明反向之间的关系转录和单一核心涂层；（3）调查宿主对HIV-1脱涂的调节因素，例如NUP358和CPSF6。这些研究采用了遗传的结合，预计生化和高级成像工具将为您提供关键见解活细胞中HIV-1解涂的动力学和调节。