Tracking single HIV viruses during infection host cells using live cell TIRF

HIV通过几个步骤进入宿主细胞，包括形成进入复合物、融合过程和向细胞核迁移以进行病毒复制。 病毒包膜gp 41-gp 120异二聚体结合成gp 41/gp 120三聚体，在病毒表面形成刺突。 结构研究表明，成熟的HIV颗粒含有10-15个刺突，每个刺突形成直径为10.5 nm的球状突起。 CD 4与gp 120的结合导致尖峰的构象变化，使得gp 120与趋化因子受体相互作用，从而完成进入复合物的形成。 gp 120与CD 4和趋化因子受体的连接引发进一步的结构变化，使gp 41插入靶细胞膜，从而引发病毒包膜和宿主细胞膜之间的融合。 融合后，含有病毒的囊泡通过皮层肌动蛋白屏障向细胞核迁移。 在巨噬细胞或T细胞中，肌动蛋白细胞骨架的重组是自发的和稳健的，皮质肌动蛋白对HIV迁移几乎没有抵抗力。 因此，似乎导致肌动蛋白解聚的趋化因子受体信号传导对于HIV感染不是必需的。 然而，在静息T细胞中，已经证明CXCR 4信号传导介导的皮质肌动蛋白基质的解聚对于HIV复制是必需的。 我们将使用相同的成像装置对假HIV病毒诱导的活细胞中皮质肌动蛋白的重组进行成像。 将Cy 5标记的假病毒置于盖玻片表面，并将静息CD 4 T细胞置于病毒顶部。 利用延时实验，我们将真实的捕捉到HIV侵入过程中，病毒触发的皮层肌动蛋白的变化。 为了测试细胞是否可以通过稳定其皮质肌动蛋白来阻止艾滋病毒进入，我们将首先用Jasplakinaldehyde（一种F-肌动蛋白稳定剂）处理细胞，然后使用延时电影跟踪艾滋病毒。 利用我们开发的软件包对病毒的运动进行分析，获得扩散常数和运动类型等参数。 这些动态信息对于更完整地理解HIV进入和皮层肌动蛋白重组机制至关重要。

HIV enters host cells through several steps, including the formation of an entry complex, the fusion process, and migration toward nucleus for viral replication. The viral envelop gp41-gp120 heterodimers associate into gp41/gp120 trimers to form spikes on the viral surface. Structural studies suggest that a mature HIV particle contains 10-15 spikes each forming a knob with a diameter of 10.5 nm. Binding of CD4 to gp120 leads to conformational changes in a spike that allows gp120 to interact with a chemokine receptor, which completes the formation of an entry complex. Ligation of gp120 to CD4 and a chemokine receptor triggers further structural changes that allow gp41 to insert into the target cell membrane, which triggers the fusion between viral envelop and host cell membrane. Upon the fusion, vesicles that contain virus migrate through cortical actin barrier towards nucleus. In macrophages or T cells where re-organization of actin cytoskeleton is spontaneous and robust, the cortical actin presents little resistance for the HIV migration. Therefore, it appears that chemokine receptor signaling that leads to actin de-polymerization is not essential for HIV infection. In resting T cells, however, it has been demonstrated that CXCR4 signaling mediated de-polymerization of cortical actin matrix is essential for HIV replication. We will image pseudo HIV virus-induced reorganization of cortical actin in live cells using the same imaging setup. Pseudo viruses labeled by Cy5 will be placed on the surface of the cover glass, and resting CD4 T cells will be placed on top of the viruses. Using time-lapse experiments, we will capture the virus-triggered changes of cortical actin during HIV entry process in real time. To test whether a cell can prevent HIV entry by stabilizing its cortical actin, we will first treat cells with jasplakinolide, an F-actin stabilizing agent, and then track HIV viruses using time-lapse movies. The movement of viruses will be analyzed by our developed software package to obtain parameters, such as diffusion constants and types of the movement. Such dynamic information is crucial for a more complete understanding of HIV entry and mechanism of reorganization of cortical actin.