HIV gp120-induced Interaction between CD4 and CCR5 Requires Cholesterol-rich Microenvironments Revealed by Live Cell Fluorescence Resonance Energy Transfer Imaging

Abstract

Binding of the human immunodeficiency virus (HIV) envelope gp120 glycoprotein to CD4 and CCR5 receptors on the plasma membrane initiates the viral entry process. Although plasma membrane cholesterol plays an important role in HIV entry, its modulating effect on the viral entry process is unclear. Using fluorescence resonance energy transfer imaging, we have provided evidence here that CD4 and CCR5 localize in different microenvironments on the surface of resting cells. Binding of the third variable region V3-containing gp120 core to CD4 and CCR5 induced association between these receptors, which could be directly monitored by fluorescence resonance energy transfer on the plasma membrane of live cells. Depletion of cholesterol from the plasma membrane abolished the gp120 core-induced associations between CD4 and CCR5, and reloading cholesterol restored the associations in live cells. Our studies suggest that, during the first step of the HIV entry process, gp120 binding alters the microenvironments of unbound CD4 and CCR5, with plasma membrane cholesterol required for the formation of the HIV entry complex.Entry of human immunodeficiency virus-1 (HIV-1) 2 into cells requires the formation of entry complexes involving the viral envelope glycoprotein gp120 and the target cell receptors CD4 and either CCR5 or CXCR4. CCR5 and CXCR4 are G-protein-coupled chemokine receptors that play critical roles in immune responses (1-4). Considerable effort has been focused on the development of therapeutics targeting chemokine receptors to block HIV entry (5, 6). A major problem in developing safe and effective co-receptor inhibitors is the risk of interfering with chemokine receptor signaling, thereby causing harmful side effects. Understanding the molecular mechanisms underlying chemokine function in HIV infection will hopefully provide a foundation for designing and screening drugs that offer strong and long lasting HIV-inhibitory function but have little effect on the physiology of the homeostatic chemokine system. In the past years, extraordinary progress has been made in solving the structures of gp120 and CD4 and in demonstrating that chemokines and small derivative molecules block HIV infection (4, 6 -11). Yet, the details regarding gp120-induced formation of HIV entry complexes in the context of live cells still need to be resolved.The plasma membrane of eukaryotic cells consists of a complex assembly of various lipids and proteins that are distributed in regions of distinct lipid microenvironments known as lipid or non-lipid raft microdomains (12-17). Lipids in rafts possess long and saturated fatty acyl chains and are organized in a tightly packed, liquid-ordered manner, whereas non-lipid raft microdomains contain shorter, unsaturated fatty acyl chains and are in a loosely packed, disordered state (12-17). Lipid rafts are defined as microdomains that are enriched with cholesterol, glycosphingolipids, and sphingomyelin and are often isolated in detergent-resistant membrane fractions. Previous studies draw different conc...

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