Role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells

Abstract

In this study we examined the effects of target membrane cholesterol depletion and cytoskeletal changes on human immunodeficiency virus type 1 (HIV-1) Env-mediated membrane fusion by dye redistribution assays. We found that treatment of peripheral blood lymphocytes (PBL) with methyl-beta-cyclodextrin (MbetaCD) or cytochalasin reduced their susceptibility to membrane fusion with cells expressing HIV-1 Env that utilize CXCR4 or CCR5. However, treatment of human osteosarcoma (HOS) cells expressing high levels of CD4 and coreceptors with these agents did not affect their susceptibility to HIV-1 Env-mediated membrane fusion. Removal of cholesterol inhibited stromal cell-derived factor-1alpha- and macrophage inflammatory protein 1beta-induced chemotaxis of both PBL and HOS cells expressing CD4 and coreceptors. The fusion activity as well as the chemotactic activity of PBL was recovered by adding back cholesterol to these cells. Confocal laser scanning microscopy analysis indicated that treatment of lymphocytes with MbetaCD reduced the colocalization of CD4 or of CXCR4 with actin presumably in microvilli. These findings indicate that, although cholesterol is not required for HIV-1 Env-mediated membrane fusion per se, its depletion from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion. Furthermore, our results suggest that coreceptor clustering may occur in microvilli that are supported by actin polymerization.

Figures

FIG. 1.

HIV-1 Env-mediated fusion with PBL and HOSCD4X4R5 cells. Fusion activity was monitored as described in Materials and Methods by using HeLa cells infected with vaccinia virus vectors which express gp120-gp41 from a CXCR4-utilizing isolate (IIIB, vPE16) and a CCR5-utilizing isolate (Ba-L, vCB43). (A) Gray bars, untreated PBL; black bars, PBL treated with 10 mM MβCD for 30 min at 37°C; white bars, MβCD-treated PBL loaded with 75 μg of cholesterol/ml complexed with MβCD. (B) HIV-1IIIB (gray bars) and HIV-1Ba-L (black bars) were expressed in HeLa cells, and fusion with HOSCD4X4R5 cells treated with different amounts of cyclodextrin (at 37°C and for 30 min) was monitored as described in Materials and Methods.

FIG. 2.

Migration of PBL and HOSCD4X4R5 cells in response to chemokines. Different concentrations of SDF-1α (top panels) or MIP-1β (bottom panels) were placed in the lower wells of the chemotaxis chamber; cells were placed in the upper wells, which were separated from the lower wells by a polycarbonate filter. The results are expressed as chemotaxis index representing the fold increase of migrating cells in response to chemokines over the response to control medium. Significant cell migration (P < 0.05) was detected with 10 ng of chemoattractant/ml. Gray bars, untreated cells; black bars, cells treated with 10 mM MβCD for 30 min at 37°C; white bars, MβCD-treated cells loaded with 75 μg of cholesterol/ml complexed with MβCD. (A) PBL; (B) HOSCD4X4R5.

FIG. 3.

HIV-1 Env-mediated fusion of target cells with different surface densities of CCR5. The figure shows the fusion of HIV-1Ba-L-expressing HeLa cells with HeLa cells expressing high (J53, gray bars) and low (J10, black bars) levels of CCR5. The treatment with MβCD was performed at 37°C for 30 min. Expression levels of CD4 and CCR5 are summarized in Table 1. The fusion activity was monitored as described in Materials and Methods.

FIG. 4.

Effect of Env expression on HIV-1 Env-mediated fusion. (A) Fusion of HIV-1IIIB-expressing HeLa cells (gray and left diagonally striped bars) with vPE16 and of TF228 cells (black and right diagonally striped bars) with HeLa cells expressing high (JC, gray and black bars) and low (RC, striped bars) levels of CD4 (Table 1 shows levels of expression of CD4 and coreceptors). The treatment with MβCD was performed at 37°C for 30 min. The use of vaccinia virus recombinant resulted in five-times-higher Env expression than that of the TF228 cells. (B) Inhibition of the fusion of TF228 cells with JC HeLa cells as a function of the time of coincubation. JC cells were treated for 30 min at 37°C with 15 mM MβCD. Upon treatment, MβCD was washed and the JC cells were incubated in PBS until their coincubation with the TF228 cells in PBS. The TF228 cells were added to the different wells at different time points starting with the longer kinetics so that the time of incubation of the MβCD-treated cells in PBS would be the same at the end of each kinetic point. The fusion activity was monitored as described in Materials and Methods.

FIG. 5.

HIV-1 receptors in DRMs. Primary lymphocytes (5 × 108 cells) were kept at 37°C, left untreated (−) or incubated for 3 min with gp120 (10 μg/ml) (+), and then subjected to a 1% Triton X-100 sucrose gradient as described in Materials and Methods. Twelve fractions were collected and quantitated by a protein assay. Equal protein amounts (4 μg for detection of CD4 [A], 100 μg for detection of CXCR4 [B], and 8 μg for detection of Zap-70 [C]) of each fraction were resolved by SDS-PAGE and subjected to immunoblot analysis with anti-CD4, anti-CXCR4, and anti-Zap-70 antibodies as indicated. DRMs are represented by fractions 4 to 7 whereas soluble proteins appear in fractions 9 to 11.

FIG. 6.

Effect of cholesterol removal on colocalization of CD4 or CXCR4 with F actin. CEM cells were left untreated (top panels) or treated with 10 mM MβCD at 37°C for 30 min (bottom panels). The cells were stained with anti-CD4 (A) or with anti-CXCR4 (B) followed by Alexa Fluor 594-conjugated secondary (Fab)2 antibodies (red) as described in Materials and Methods. Cells were then fixed, treated with 0.5% Triton X-100, labeled with Alexa Fluor 488-conjugated phalloidin (green) at 37°C for 30 min, and examined by confocal microscopy as described in Materials and Methods. Bar, 5 μm. The white color on the images indicates CD4-actin or CXCR4-actin colocalization.

FIG. 7.

Effects of cytochalasin on HIV-1 Env-mediated fusion. Fusion activity was monitored as described in Materials and Methods between HeLa cells infected with a vaccinia virus vector which expresses gp120-gp41 from a CXCR4-utilizing isolate (IIIB, vPE16) and PBL or HOSCD4X4R5 as indicated. Gray bars, untreated cells; black bars, cells treated with 10 mM MβCD for 30 min at 37°C; white bars, cells treated with 10 μM cytochalasin B. PBMC, peripheral blood mononuclear cells.

FIG. 8.

Model for the effect of the cell surface disposition of CD4 and coreceptor on the triggering of HIV-1 Env-mediated fusion. CD4 is located on the tip of a microvillus whereas CXCR4 or CCR5 is located more toward the base (64). Interaction of gp120 on HIV-1 or on HIV-1 Env-expressing cells with CD4 has two consequences: (i) triggering of conformational changes in Env that expose binding sites for coreceptor (70, 74) and (ii) signaling events that lead to rearrangements of actin in the microvilli (26). As a result the triggered Env is able to engage coreceptor, leading to gp41 six-helix bundle formation and fusion (23). Treatment of cells with MβCD or inhibitors of F-actin polymerization disrupts the microvilli. As a consequence CD4-triggered Env becomes less capable of engaging coreceptors on cells expressing low levels of coreceptor. However, the effect of these treatments can be overcome in cells expressing levels of coreceptor high enough to engage CD4-triggered Env.