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

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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.