Single virions to study assembly of HIV-1

描述（由申请人提供）：在培养细胞系和原代细胞中，HIV-1组装的生产位点是质膜。这一结论是基于观察到新合成的Gag首先出现在质膜上，然后才在核内体中检测到；抑制内噬作用可阻断病毒粒子在细胞器内的出现，而不影响细胞外颗粒的产量。HIV-1在质膜上的组装使其可以使用全内反射荧光显微镜（TIR-FM）成像，这是本提案中大部分实验所基于的技术。这项技术已被用于观察HIV-1单个病毒粒子的组装过程，以及单个分子或HIV-1基因组二聚体的运动和包装。该技术已被用于证明病毒粒子在6-20分钟的时间内在质膜上积累。基因组在Gag募集之前立即被募集到质膜上。相比之下，在Gag募集的最后阶段，escrt只会被短暂地募集几十秒和几十个分子。aaa - atp酶Vps4在几秒钟后被招募。超分辨率光学显微镜增加了ESCRT招募是连接新生病毒粒子和母细胞的颈部的信息。该项目的长期目标是确定、表征并最终了解HIV-1和相关逆转录病毒的生物发生步骤。我们想了解病毒成分相互作用以及与宿主成分相互作用时的动力学。基于成像的方法允许检查分子的动力学和定位，在此期间可能无法通过生化技术。主要的重点是病毒蛋白Gag的动力学和定位，宿主分子的动力学和定位，然后是病毒和宿主分子的相对动力学和定位。

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.