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

Nilgun Isik,Jimmy Chim,Jun Fang,Ling Yi,Tian Jin

doi:10.1074/jbc.m607302200

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.

Highlights

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
Using detergent insolubility or immunostaining with fluorescence microscopy, several studies suggest that CD4 and CCR5 receptors localize in lipid rafts, and this localization is important for ligand binding, receptor signaling, and the formation of HIV entry complexes (18 –20)
In contrast to these reports, other reports suggest that the localization of CD4 and CCR5 in the lipid raft microdomains is not required for HIV infection and conclude that HIV entry into T-cells does not depend on the localization of CD4 and CCR5 to cholesterol-enriched, detergent-resistant membrane microdomains [20, 21]

Summary

EXPERIMENTAL PROCEDURES

Chemicals and Reagents—pEYFP- N1 and pECFP-N1 were purchased from Clontech (Palo Alto, CA). The HEK293T cells were transfected or co-transfected with the plasmids encoding CD4-YFP and/or CCR5-CFP mediated by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Two bands were detected by anti-GFP antibodies in the cells expressing CCR5-CFP. B, confocal images of living cells expressing membrane-localized CCR5-CFP (cyan). C, fusion protein of CCR5-CFP was detected by Western blotting in whole cell lysate using an anti-GFP antibody. E, confocal images of living cells expressing membrane-localized CD4-YFP (yellow). CCR5-CFP fusion protein, we examined the ligand-induced Ca2ϩ response in the CCR5-CFP-expressing cells (Fig. 2). We imaged a fluorescence intensity change of Fluo-4 (a fluorescent calcium indicator) trigged by MIP1␣ (a ligand for CCR5). Upon the addition of MIP1␣ to the cell chamber, the green fluorescence signal transiently increased in the cytosol, indicating that MIP1␣ induced changes in Ca2ϩ concentration (Fig. 2). No apparent increase in green fluorescence signal was observed after MIP1␣ stimula-

RESULTS

Findings

DISCUSSION