To remove cholesterol from cells, we used the cholesterol-solubilizing agent methyl-β-cyclodextrin (MβCD), which stimulates cholesterol efflux from cells 12. Figure 1 shows that incubation of NIH3T3 cells with 10 mM MβCD for 30 min at 37°C reduced cholesterol levels of the cells to about 22% of untreated controls. There was no significant effect on cell viability (not shown) or surface expression of CD4 and coreceptors 13. Figure 2 shows that fusion mediated by CD4-independent HIV-1 Env was reduced to about 30% of untreated controls for both 8x and 8xV3BAL Envs, irrespective of the presence of CD4 in the target membrane. When these cells were replenished with cholesterol by incubation with pre-formed cholesterol- MβCD complexes, HIV-1 env-mediated fusion was fully recovered. Interestingly, in spite of the fact that cholesterol levels were only replenished to 50% of untreated control (Figure 1), HIV-1 Env-mediated fusion was fully recovered (Figure 2). This observation is consistent with the notion that the dependence of membrane organization on cholesterol is not linear, but rather that there is a critical concentration of cholesterol below which phase separation of domains occurs 14.

To modulate shingolipid metabolism, we used an inhibitor of GlcCer synthase, PPMP 15. The effects of such treatment was monitored by observing changes in the expression of cell surface glycosphingolipids by flow cytometry following the staining of the cells with anti-glycosphingolipid antibodies 16. In accordance with previous studies we observed a 4–5 reduction in GM3 levels following treatment of the cells with 10 μM PPMP for 48 h (data not shown). Phospholipid and cholesterol composition of the cells were unchanged following PPMP treatment as shown by our previously studies 17. Figure 3 shows the cell surface expression levels of CD4, CXCR4 and CCR5 on control and PPMP treated cells. Although there was no significant change in cell surface expression of CD4 or CXCR4, the level of CCR5 expression was reduced by about 50% following treatment of these cells with PPMP consistent with our previous findings 17.

Cells expressing the 8x and 8xV3BAL variants of HIV-1_IIIB were incubated at different times with NIH3T3 cells bearing CXCR4 and CCR5, respectively, with or without CD4. Fusion was monitored before and after treatment of the cells with PPMP. Although the Env constructs we used did not require CD4 for fusion activity it appeared that, in the case of the CXCR4 construct, presence of CD4 on the target cells enhanced the fusion reaction (Figure 4), presumably as a result of better attachment or more robust conformational changes, In the case of the CCR5 construct, presence of CD4 on the target cell did not speed up the fusion reaction significantly (Figure 5). However, there was a significant difference in fusion mediated by the CCR5 construct at the different time points between untreated and PPMP-treated target cells (Figure 5) as indicated by the p values determined by paired student t tests. However, this reduction in fusion activity can be explained by the reduced levels of cell surface CCR5 (Figure 3). PPMP did not have a significant effect on fusion mediated by the CD4-independent CXCR4 construct as indicated by the p values determined by paired student t tests (Figure 4). Collectively, these data indicate that PPMP-treatment does not affect the one-step fusion pathway mediated by HIV-1 Env.

Nguyen and coworkers showed that removal of cholesterol from the cell membrane results in loss in ligand binding which is likely due to conformational changes in CXCR4 18 or CCR5 19. In accordance with those results, Figure 6 shows that Ca²⁺ mobilization in NIH3T3 cell lines expressing CXCR4 or CCR5 in response SDF-1α or MIP-1β, respectively, was inhibited following treatment of the cells with MβCD. If treatment of these cell lines with PPMP has a similar effect on ligand binding, we would expect Ca²⁺ mobilization in NIH3T3 cell lines expressing CXCR4 or CCR5 in response SDF-1α or MIP-1β, respectively, to be inhibited following treatment with PPMP. By contrast, treatment of these cells with PPMP had no effect on chemokine receptor signalling (Figure 6). Thus, the cholesterol effect can be explained by changes in chemokine receptor disposition that leads to engagement with HIV-1 Env.

Phycoerythrin (PE)-labeled Mabs against CD4, CXCR4 and CCR5 and their isotype controls were obtained from Pharmingen (San Diego, CA). The anti-GM3 Mab GMR6 was obtained from Seikagaku America (Falmouth, MA) and Cy3-conjugated anti-mouse Fab from Jackson Immunochemicals (West Grove, PA). Methyl-β-cyclodextrin (MβCD) and cholesterol/MβCD complexes purchased from Sigma (St. Louis, MI), and 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP) from Matreya, Inc. (Pleasant Gap, PA). Cholesterol in total cell lysate was determined using the Cholesterol Oxidase kit (Wako Chemicals USA Inc, Richmond, VA). The cytoplasmic dyes 5-chloromethylfluorescein diacetate (CMFDA), 5- and 6-([(4-chloromethyl)benzoyl]-amino)tetramethyl-rhodamine (CMTMR) and Fura-2 AM were from Molecular Probes (Eugene, OR). The chemokine receptor ligands SDF1-α and MIP1-β were from Peprotech (Rocky Hill, NJ). All other biochemicals used were of the highest purity available and were obtained from regular commercial sources.

NIH 3T3 cells constitutively expressing CD4 and/or CXCR4, and NIH 3T3 cells constitutively expressing CD4 and/or CCR5, were kindly provided by Dr. Dan Littman (New York University, New York). HIV-1 Env 8x and 8xV3BAL 36, kindly provided by Dr. Robert Doms, University of Pennsylvania, Philadelphia, were expressed on the surface of NIH3T3 cells by transfection using the vaccinia T7-polymerase (vTF7-3), which was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH from Drs Tom Fuerst and Bernard Moss.

The fluorescence of cells stained with PE-conjugated Mabs against CD4, CXCR4 and CCR5 was compared with the appropriate PE-conjugated isotype controls. Cell labeling with PE-conjugated Mabs was done in the presence of 2% serum, after which cells were washed and fixed with 1% paraformaldehyde. The analysis was performed on a FACStar plus flow cytometer. GM3 levels were monitored by immunofluorescence using the anti-GM3 Mab (GMR6) and a Cy5-stained antimouse Fab.

To express 8x or 8x-V3BaL gp120-gp41, NIH3T3 cells plated on T75 flasks were incubated with the vaccinia virus vTF7-3 (5 M.O.I.) for 1–2 hrs at 37°C. Subsequently, the cells were transfected with 15 μg DNA (psp73.8x or psp73.8x.V3BaL using lipofectamine (Invitrogen, Inc.) in 3 ml DMEM (without serum) and incubations were continued for 4–6 hrs at 37°C. DMEM containing 10% FBS + antibiotics (D10) was added and cells were incubated for additional 16–20 hours. For the fusion assay, cells were harvested using EDTA-based cell dissociation buffer (Life Technologies, Inc.) and labeled with 10 μM CMFDA following manufacturer's directions. The CMFDA-labeled HIV-1 Env-expressing cells (2 × 10⁵ per well) were added to the same number of CMTMR-labeled NIH3T3 targets expressing CD4 and/or cognate co-receptors and the samples were incubated for 4–10 hrs at 37°C. Dye redistribution as a result of fusion was monitored microscopically as described previously 37. The extent of fusion was calculated as: Percent Fusion = 100 × number of cells positive for both dyes/number of bound cells positive for CMTMR.

Intracellular [Ca²⁺] mobilization was measured by incubating 2 × 10⁷ cells/ml in loading medium (DMEM containing 10% FCS and 2 mM glutamine) with 7 mM Fura-2 AM for 45 min at room temperature. The dye-loaded cells were washed and resuspended in saline buffer (138 mM NaCl, 6 mM KCl, 1 mM CaCl2, 10 mM HEPES, 5 mM glucose, and 0.1% BSA, pH 7.4) at a density of 0.5 × 10⁶/ml. The cells were then transferred into quartz cuvettes (1 × 10⁶ cells in 2 ml saline buffer), which were placed in a fluorescence spectrometer (Perkin-Elmer, Beaconsfield, U. K.). Chemokines were added in a volume of 20 ml to each cuvette. The intensity of the fluorescence was measured as the ratio at 340 and 380 nm wavelengths and calculated using a FL WinLab program (Perkin-Elmer).