Analysis of the lipid composition of influenza virus–infected cells provides support for the membrane raft-based biogenesis model.

We document a new dimension of surface recognition in which communication is controlled through the collective behavior of lipids. Membrane cholesterol induces a tilt in glycolipid receptor headgroup, resulting in loss of access for ligand binding. This property appears to organize erythrocyte blood group presentation and glycolipid receptor function during the activation of sperm fertility, suggesting that lipid 'allostery' is a means to regulate membrane recognition processes.

The lung is a preferential (Gb(3)) "sink" for VT1, which explains the relatively slower clearance of VT2 and subsequent increased VT2 renal targeting and VT2 mortality in this animal model.

The glycosphingolipid globotriaosyl ceramide, (Galα1‐4Galß1‐4 glucosyl ceramide‐Gb3) also known as CD77 and the Pk blood group antigen, is bound by both verotoxins and by the HIV adhesin, gp120. Gb3 plays an important receptor role in VT induced hemolytic uremic syndrome (HUS) and HIV infection. The organization of glycolipids, including Gb3, into lipid rafts is central to both pathologies. The fatty acid heterogeneity within the Gb3 lipid moiety plays a central role in assembly within such ordered domains. Differential binding of verotoxins and gp120 to such Gb3 isoforms in model and cell membranes indicates a significant role in the eventual pathogenic outcome. HUS may provide the first example whereby membrane Gb3 organization provides a predictor for tissue selective in vivo pathology.

Although verotoxin-1 (VT1) and verotoxin-2 (VT2) share a common receptor, globotriaosyl ceramide (Gb(3)), VT2 induces distinct animal pathology and is preferentially associated with human disease. Moreover VT2 cytotoxicity in vitro is less than VT1. We therefore investigated whether these toxins similarly traffic within cells via similar Gb(3) assemblies. At 4 degrees C, fluorescent-VT1 and VT2 bound both coincident and distinct punctate surface Gb(3) microdomains. After 10 min at 37 degrees C, similar distinct/coincident micropunctate intracellular localization was observed. Most internalized VT2, but not VT1, colocalized with transferrin. After 1 h, VT1 and VT2 coalesced during retrograde transport to the Golgi. During prolonged incubation (3-6 h), VT1, and VT2 (more slowly), exited the Golgi to reach the ER/nuclear envelope. At this time, VT2 induced a previously unreported, retrograde transport-dependent vacuolation. Cell surface and intracellular VT1 showed greater detergent resistance than VT2, suggesting differential 'raft' association. >90% (125)I-VT1 cell surface bound, or added to detergent-resistant cell membrane extracts (DRM), was in the Gb(3)-containing sucrose gradient 'insoluble' fraction, whereas only 30% (125)I-VT2 was similarly DRM-associated. VT1 bound more efficiently to Gb(3)/cholesterol DRMs generated in vitro. Only VT1 binding was inhibited by high cholesterol/Gb(3) ratios. VT2 competed less effectively for (125)I-VT1/Gb(3) DRM-binding but only VT2-Gb(3)/cholesterol DRM-binding was augmented by sphingomyelin. Differential VT1/VT2 Gb(3) raft-binding may mediate differential cell binding/intracellular trafficking and cytopathology.

Glycosphingolipids (GSLs) accumulate in cholesterol-enriched cell membrane domains and provide receptors for protein ligands. Lipid-based "aglycone" interactions can influence GSL carbohydrate epitope presentation. To evaluate this relationship, Verotoxin binding its receptor GSL, globotriaosyl ceramide (Gb 3 ), was analyzed in simple GSL/cholesterol, detergent-resistant membrane vesicles by equilibrium density gradient centrifugation. Vesicles separated into two Gb 3/ cholesterolcontaining populations. The lighter, minor fraction (<5% total GSL), bound VT1, VT2, IgG/IgM mAb anti-Gb 3 , HIVgp120 or Bandeiraea simplicifolia lectin. Only IgM anti-Gb 3 , more tolerant of carbohydrate modification, bound both vesicle fractions. Post-embedding cryo-immuno-EM confirmed these results. This appears to be a general GSL-cholesterol property, because similar receptor-inactive vesicles were separated for other GSLprotein ligand systems; cholera toxin (CTx)-GM1, HIVgp120-galactosyl ceramide/sulfatide. Inclusion of galactosyl or glucosyl ceramide (GalCer and GlcCer) rendered VT1-unreactive Gb 3 /cholesterol vesicles, VT1-reactive. We found GalCer and GlcCer bind Gb 3 , suggesting GSL-GSL interaction can counter cholesterol masking of Gb 3 . The similar separation of Vero cell membrane-derived vesicles into minor "binding," and major "non-binding" fractions when probed with VT1, CTx, or anti-SSEA4 (a human GSL stem cell marker), demonstrates potential physiological relevance. Cell membrane GSL masking was cholesterol-and actin-dependent. Cholesterol depletion of Vero and HeLa cells enabled differential VT1B subunit labeling of "available" and "cholesterol-masked" plasma membrane Gb 3 pools by fluorescence microscopy. Thus, the model GSL/cholesterol vesicle studies predicted two distinct membrane GSL formats, which were demonstrated within the plasma membrane of cultured cells. Cholesterol masking of most cell membrane GSLs may impinge many GSL receptor functions.

These results suggest that adamantylGb3 may provide a new basis for blocking HIV infections, irrespective of HIV envelope/chemokine co-receptor preference or resistance to other therapeutics.

Several human histo-blood groups are glycosphingolipids, including P/P1/P k . Glycosphingolipids are implicated in HIVhost-cell-fusion and some bind to HIVgp120 in vitro. Based on our previous studies on Fabry disease, where P k accumulates and reduces infection, and a soluble P k analog that inhibits infection, we investigated cell surface-expressed P k in HIV infection. HIV-1 infection of peripheral blood-derived mononuclear cells (PBMCs) from otherwise healthy persons, with blood group P 1 k , where P k is overexpressed, or blood group p, that completely lacks P k , were compared with draw date-matched controls. Fluorescenceactivated cell sorter analysis and/or thin layer chromatography were used to verify P k levels. P 1 k PBMCs were highly resistant to R5 and X4 HIV-1 infection. In contrast, p PBMCs showed 10-to 1000-fold increased susceptibility to HIV-1 infection. Surface and total cell expression of P k , but not CD4 or chemokine coreceptor expression, correlated with infection. P k liposome-fused cells and CD4 ؉ HeLa cells manipulated to express high or low P k levels confirmed a protective effect of P k . We conclude that P k expression strongly influences susceptibility to HIV-1 infection, which implicates P k as a new endogenous cell-surface factor that may provide protection against HIV-1 infection. (Blood. 2009;113:4980-4991)