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

• 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
• 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

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.

在病毒-细胞融合后，锥形HIV-1衣壳的降解是 造成终身感染HIV-1进入的这一步，被称为脱膜，是至关重要的， 不太了解。我们最近开发了一种新的策略来可视化HIV-1的脱壳， 是基于荧光标记的寡聚形式的captain结合宿主蛋白亲环素A （CypA-DsRed）。CypA-DsRed通过高亲合力特异性包装成病毒粒子， 与HIV-1衣壳结合，不损害感染性。这个探测器仍然与 在病毒-细胞融合后具有核心，并且在脱壳时释放。支持这一观点的是我们的 发现来自单个融合后核心的CypA-DsRed损失率受到以下因素的调节： 影响核心稳定性的突变，并通过逆转录加速。CypA-DsRed 基于成像分析揭示了HIV-1脱壳的双相动力学，具有大量核心 融合后不久脱落衣壳蛋白。该试验还显示， HeLa衍生细胞和原代人巨噬细胞中的未包被表型。我们提出 目的：（1）通过功能和结构描述CypA-DsRed与HIV-1核心的相互作用 CypA-DsRed/衣壳复合物的研究;（2）阐明了逆转录病毒与细胞凋亡之间的关系。 转录和单核脱壳;（3）研究宿主对HIV-1脱壳的调控 Nup 358和CPSF 6等因素。这些研究采用了基因、 生物化学和先进的成像工具，预计将提供关键的见解， 动态和调节HIV-1在活细胞中的脱壳。