Single virions to study assembly of HIV-1

 描述(申请人提供)：HIV-1组装的产生部位是质膜，在培养的细胞系和原代细胞中都是如此。这一结论的基础是，新合成的Gag首先出现在质膜上，后来才在内小体中检测到，以及证明抑制内吞作用阻止了内部细胞器中病毒粒子的出现，而不影响细胞外颗粒的产量。HIV-1在质膜上的组装使该组装可以使用全内反射荧光显微镜(TIR-FM)进行成像，这是本建议中的大部分实验所基于的技术。这项技术已被用于可视化HIV-1的单个病毒粒子组装以及HIV-1基因组的单个分子或二聚体的移动和包装。这项技术已经被用来证明病毒粒子在质膜上积累了6-20分钟。基因组在GAG招募之前立即被招募到质膜上。相比之下，ESCRT只被招募了几十秒，在GAG招募的最后一刻瞬时招募了数十个分子。几秒钟后，AAA-ATPase Vps4就被招募了。超分辨率光学显微镜补充了ESCRT招募是在颈部连接新生的病毒粒子和母细胞的信息。该项目的长期目标是确定、描述并最终了解艾滋病毒-1和相关逆转录病毒生物发生的步骤。我们想要了解病毒成分相互作用以及与宿主成分相互作用时的动态。基于成像的方法允许检查分子的动力学和局部化，在此期间可能无法通过生化技术获得。主要焦点将集中在病毒蛋白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.