Single virions to study assembly of HIV-1

单一病毒体研究 HIV-1 的组装

• 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
• 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

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）：HIV-1 组装的产生位点是质膜，无论是在培养细胞系还是原代细胞中。这一结论基于以下观察结果：新合成的 Gag 首先出现在质膜上，随后才在内体中检测到，并且证明抑制内吞作用会阻止病毒粒子在内部细胞器中的出现，而不影响细胞外颗粒的产量。 HIV-1 在质膜上的组装使得可以使用全内反射荧光显微镜（TIR-FM）对组装进行成像，该技术是本提案中大部分实验的基础。该技术已用于可视化 HIV-1 单个病毒粒子的组装以及 HIV-1 基因组单个分子或二聚体的运动和包装。该技术已被用来证明病毒颗粒在质膜上积累了 6-20 分钟的时间。基因组在 Gag 募集之前立即被募集到质膜上。相比之下，ESCRT 的募集时间仅为数十秒，并且在 Gag 募集的最后阶段短暂地募集了数十个分子。 AAA-ATP酶，Vps4，在几秒钟后被招募。超分辨率光学显微镜添加了这样的信息，即 ESCRT 招募位于将新生病毒体与母细胞连接起来的颈部。该项目的长期目标是识别、表征并最终了解 HIV-1 和相关逆转录病毒的生物发生步骤。我们希望了解病毒成分之间以及与宿主成分相互作用时的动态。基于成像的方法可以检查分子的动力学和定位，而这可能是通过生化技术无法实现的。主要关注点是病毒蛋白 Gag 的动力学和定位、宿主分子的动力学和定位，以及病毒和宿主分子的相对动力学和定位。