Using FRET to Probe the Spatial Distributions of CD4, CXCR4 and CCR5 on Membrane

HIV通过病毒包膜（Env）糖蛋白与细胞表面蛋白CD4和趋化因子受体CXCR4或CCR5的连续相互作用进入细胞。病毒包膜gp120-gp41异源二聚体以三聚体形式结合，在病毒表面形成尖刺。结构研究表明，一个成熟的HIV颗粒包含72个尖刺，两个尖刺之间的距离为21-22 nm，一个尖刺形成一个直径为14 nm的旋钮。CD4与gp120结合导致尖峰的构象变化，使gp120能够与趋化因子受体相互作用。gp120与CD4和趋化因子受体的连接引发进一步的结构变化，使gp41能够插入目标细胞膜。在目前的模型中，一个gp120与一个CD4和一个趋化因子受体相互作用。免疫电镜证据表明，在接触HIV之前，CD4、CXCR4和CCR5各自形成独立的微团簇，它们相隔约10 nm的距离。基于结构和免疫电镜研究，我们估计当HIV进入复合物形成时，CD4和趋化因子受体之间的距离在10nm以内。我们支持的假设是，当gp120三聚体结合CD4分子时，gp120-CD4复合物诱导趋化因子受体与三聚体结合形成高产的HIV进入复合物。免疫染色显示gp120诱导CD4和CXCR4或CCR5共定位。然而，由于免疫染色的分辨率限制，CD4与趋化因子受体之间的时空排列和物理相互作用尚不清楚。

HIV enters cells through sequential interactions of the viral envelope (Env) glycoprotein with the cell surface protein CD4 and a chemokine receptor CXCR4 or CCR5. The viral envelope gp120-gp41 heterodimers associate in a trimer to form spikes on the viral surface. Structural studies suggest that a mature HIV particle contains 72 spikes and the distance between two spikes is 21-22 nm, and a spike forms a knob with a diameter of 14 nm. Binding of CD4 to gp120 leads to conformational changes in a spike that allows the gp120 to interact with a chemokine receptor. Ligation of gp120 to CD4 and a chemokine receptor triggers further structural changes that allow the gp41 to insert into the target cell membrane. In the current model, one gp120 interacts with one CD4 and one chemokine receptor. Evidence from immunoelectron microscopy demonstrated that before contacting HIV, CD4, CXCR4 and CCR5 each form independent microclusters that are separated by a distance of about 10 nm. Based on structural and immuno-EM studies, we estimate that the distance between CD4 and the chemokine receptors is well within 10nm when a HIV entry complex is formed. Our favored hypothesis is that when gp120 trimers bind CD4 molecules, gp120-CD4 complexes induce the association of chemokine receptors with the trimers to form productive HIV entry complexes. Colocalization of CD4 and CXCR4 or CCR5 induced by gp120 has been shown using immuno-staining. However, due to resolution limitations of immuno-staining, the temporal and spatial arrangement and physical interactions between CD4 and the chemokine receptors are still unclear. We have employed fluorescence resonance energy transfer (FRET) between chimeras of CD4, CXCR4 and CCR5 fused with CFP or YFP to probe their membrane distribution and to visualize molecular interactions of CD4 and the chemokine receptors in living cells. We have also used fluorescence recovery after photobleaching (FRAP) to measure lateral diffusion of CD4 and CCR5 that each fussed with CFP or YFP in living cell membrane to probe their spatial distribution. We found that gp120 induced FRET increase between CD4-YFP and CCR5-CFP, suggesting that HIV envelope protein promotes association between CD4 and CCR5 on the cell membrane. The plasma membranes consist of a complex assembly of various lipids and proteins that are distributed in regions of distinct lipid microenvironments, known as lipid raft or non-raft microdomains. Lipid rafts are defined as microdomains that are enriched in cholesterol, glycosphingolipid and sphingomyelin. Both lipid and non-lipid raft microdomains contain multiple proteins that play critical roles in signal transduction via complex protein-protein interactions between ligands, receptors, and signaling components. To examine whether lipid raft microenvironment is essential for gp120-induced association between CD4 and CCR5, we disrupted these microenvironments on the plasma membrane by depleting cholesterol with MRCD and measured FRET between CD4-YFP and CCR5-CFP. We found interestingly that depleting cholesterol had no effect on gp120-promoted association between CD4 and CCR5. Using FRAP measurement, we showed that CD4 is more mobile than CCR5 on the plasma membrane. However, when both CD4 and CCR5 were expressed on the membrane, their mobility became similar, suggesting some interactions between these two receptors on the membrane even in the absence of gp120. We are in the process to complete FRET and FRAP experiments, which may provide insight into molecular interactions between CD4 and the chemokine receptors during HIV entry.