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Cholesterol is Essential for Chemokine Binding to Receptors, CCR5 and CXCR4: Implications for HIV Infection
D. H. Nguyen* and D. D. Taub
NIH, Baltimore, MD
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Background: The human immunodeficiency virus (HIV) utilizes CCR5 or CXCR4 as co-receptors in combination with CD4 for viral entry, which is believed to occur at cholesterol- and sphingolipid-rich membrane microdomains termed lipid rafts. However the role of cholesterol and lipid rafts on T-cell chemokine binding and signaling through chemokine receptors remains unknown.
Methods: To analyze chemokine binding we used biotinylated forms of SDF-1alpha and MIP-1beta followed by labeling with avidin-fluorescein. Hydroxypropyl-beta-cyclodextrin (BCD) was used to extract cholesterol from CEM-NKR T cells expressing both CXCR4 and CCR5 (CEM-R5). We also examined BCD effects on intracellular calcium mobilization and monoclonal antibody binding. To analyze surface localization of chemokine binding, we used immunofluorescence microscopy with cholera toxin, B subunit, binding to detect GM1 in lipid rafts.
Results: BCD treatment significantly reduced binding and signaling of MIP-1beta and SDF-1alpha in CEM-R5 cells. Reloading cells with cholesterol restored binding and signaling by both chemokines. Antibodies specific for distinct CCR5 epitopes lost their ability to bind to the cell surface after cholesterol extraction, as did 12G5, a multidomain recognizing antibody to CXCR4. The effects on antibody binding were reversed by reloading BCD-treated cells with cholesterol or fixation with paraformaldehye. Cell surface microscopy revealed that chemokine binding colocalized with GM1, while antibodies to the chemokine receptors only localized with GM1 in capped cells.
Conclusions: Together, these data demonstrate that cholesterol and lipid rafts are important for the maintenance of chemokine receptor conformation and are necessary for both the binding and function of chemokine receptors, CCR5 and CXCR4. We propose that the inhibition of HIV infection by BCD treatment of T cells may be due to a loss in binding and fusion of HIV env mediated by chemokine receptors.
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