Single virions to study assembly of HIV-1

单一病毒体研究 HIV-1 的组装

基本信息

批准号：
9459948
负责人：
SANFORD M SIMON
金额：
$ 33.48万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-05 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9459948
关键词：
ABCE1 gene ATP phosphohydrolase Affect Anisotropy Antiviral Agents Appearance Biochemical Biogenesis Biological Assay CRISPR/Cas technology Cell Line Cell membrane Cells Color DegP protease Dimensions Endocytosis Endosomes Event Fluorescence Microscopy Fluorescence Resonance Energy Transfer Genome Goals HIV HIV-1 Image Individual Infection Kinetics Label Ligase Link Location Membrane Microscopy Monitor Mothers Movement Neck Nucleotides Optics Organelles Peptide Hydrolases Play Point Mutation Population Process Production Proteins Resolution Retroviridae Role Site Structure Techniques Testing Tissues Viral Virion Virus Virus Assembly base dimer experimental study extracellular fluorophore gag Gene Products helicase monomer particle public health relevance recruit sensor spatial relationship

ABCE1 gene ATP phosphohydrolase Affect Anisotropy Antiviral Agents Appearance Biochemical Biogenesis Biological Assay CRISPR/Cas technology Cell Line Cell membrane Cells Color DegP protease Dimensions Endocytosis Endosomes Event Fluorescence Microscopy Fluorescence Resonance Energy Transfer Genome Goals HIV HIV-1 Image Individual Infection Kinetics Label Ligase Link Location Membrane Microscopy Monitor Mothers Movement Neck Nucleotides Optics Organelles Peptide Hydrolases Play Point Mutation Population Process Production Proteins Resolution Retroviridae Role Site Structure Techniques Testing Tissues Viral Virion Virus Virus Assembly base dimer experimental study extracellular fluorophore gag Gene Products helicase monomer particle public health relevance recruit sensor spatial relationship

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项目摘要

DESCRIPTION (provided by applicant): The productive site of HIV-1 assembly is the plasma membrane, both in cultured cell lines and primary cells. This conclusion is based both on the observation that newly synthesized Gag first appears at the plasma membrane, and is only detected later in endosomes, and the demonstration that inhibiting endocytosis blocks the appearance of virions in the internal organelles without affecting the yield of extracellular particles This assembly of HIV-1 at the plasma membrane make the assembly accessible to imaging using total internal reflection fluorescence microscopy (TIR-FM) a technique on which a large fraction of the experiments in this proposal are based. This technique has been used to visualize individual virions of HIV-1 as they assemble as well as the movement and packaging of individual molecules or dimers of HIV-1 genome. The technique has been used to demonstrate that the virions accumulate at the plasma membrane over a period of 6-20 minutes. The genome is recruited to the plasma membrane immediately before recruitment of Gag. In contrast, ESCRTs are recruited for only tens of seconds and tens of molecules transiently at the very end of the recruitment of Gag. The AAA-ATPase, Vps4, is recruited just seconds later. Super-resolution optical microscopy has added the information that ESCRT recruitment is to the neck that links the nascent virion to the mother cell. The long-term goal of this project is to identify, characterize and ultimately understand the steps in the biogenesis of HIV-1 and related retroviruses. We want to understand the dynamics of viral components as they interact with each other and with host components. Imaging based approaches allow the examination of both the dynamics and localization of molecules during which may be inaccessible through biochemical techniques. The main foci will be on the dynamics and localization of the viral protein Gag, the dynamics and localization of host molecules and then the relative dynamics and localization of the viral and host molecules.

描述（由申请人提供）：在培养的细胞系和原代细胞中，HIV-1组装的生产力是质膜。这一结论既基于以下观察，即新合成的堵嘴首先出现在质膜上，并且只有在后期才检测到内体中，抑制内吞作用的证明会阻止内细胞器中病毒体在内部细胞器中的出现，而不会影响质体膜膜的内部反射率的纤维外粒子的产量，而不会影响质膜膜的内部含量，这是质膜膜膜膜的组合的产量。（TIR-FM）该提案中大部分实验的技术。该技术已用于可视化HIV-1的单个病毒体，以及它们组装和包装HIV-1基因组的单个分子或二聚体的运动和包装。该技术已用于证明病毒体在6-20分钟内积聚在质膜上。募集GAG之前，该基因组将立即招募到质膜。相比之下，在募集GAG的末尾，将ESCRT募集仅数十秒和数十个分子。仅几秒钟后，AAA-ATPase VPS4被招募。超分辨率光学显微镜已将ESCRT募集的信息添加到将新生病毒粒子与母细胞联系起来的颈部。该项目的长期目标是识别，表征并最终了解HIV-1和相关逆转录病毒生物发生的步骤。我们想了解病毒组件相互交互以及与宿主组件相互作用时的动力学。基于成像的方法允许研究分子的动力学和定位，在此过程中，通过生化技术可能无法访问。主要灶将是关于病毒蛋白GAG的动力学和定位，宿主分子的动力学和定位，然后是病毒和宿主分子的相对动力学和定位。