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Membrane raft microdomains mediate lateral assemblies required for HIV-1 infection - PubMed

Membrane raft microdomains mediate lateral assemblies required for HIV-1 infection

S Mañes et al. EMBO Rep. 2000 Aug.

Abstract

HIV-1 infection triggers lateral membrane diffusion following interaction of the viral envelope with cell surface receptors. We show that these membrane changes are necessary for infection, as initial gp120-CD4 engagement leads to redistribution and clustering of membrane microdomains, enabling subsequent interaction of this complex with HIV-1 co-receptors. Disruption of cell membrane rafts by cholesterol depletion before viral exposure inhibits entry by both X4 and R5 strains of HIV-1, although viral replication in infected cells is unaffected by this treatment. This inhibitory effect is fully reversed by cholesterol replenishment of the cell membrane. These results indicate a general mechanism for HIV-1 envelope glycoprotein-mediated fusion by reorganization of membrane microdomains in the target cell, and offer new strategies for preventing HIV-1 infection.

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Figures

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Fig. 1. Membrane raft integrity is required for gp120-induced lateral association of CD4 and CXCR4. (A) Confocal images of co-patching between biotinylated CTx (a2, a5, a8) and either an anti-gp120 polyclonal antibody (a1, a4) or anti-TfR mAb (a7) in mock-transduced and CD4+ cells. Primary reagents were visualized by addition of streptavidin–Cy5 and anti-mouse or anti-rabbit Cy3 secondary antibodies. The overlay is shown (a3, a6, a9). (B) 293-CD4 (b1–b8) or PBMC (b9–b16) cells, alone (–) or incubated with gp120 (+), were co-patched with anti-gp120, biotinylated CTx, anti-TfR mAb, FITC-labeled anti-CD4 mAb, or biotinylated or FITC–anti-CXCR4 mAb as indicated, and analyzed by confocal microscopy. Only the three-color overlay is shown. (C) Co-patching with anti-gp120 (red) and anti-CD4, anti-CXCR4 or CTx (green) in CD-treated and cholesterol-replenished CD-treated 293-CD4 cells. Only the overlay image of a representative confocal section is shown. Bar: 5 µm for 293 cells and 10 µm for PBMC.

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Fig. 2. gp120 is found in the DIG fraction of the cell membrane. TX-100-insoluble membranes were isolated from gp120-treated or untreated 293-mock or 293-CD4 cells, and fractions analyzed by western blotting with antibodies to gp120, CD4 (Leu3A), CXCR4, TfR (Zymed Labs, South San Francisco, CA) and caveolin-1 (Cav; Santa Cruz Biotech., Santa Cruz, CA).

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Fig. 3. Cholesterol depletion specifically inhibits HIV-1 infection. p24 antigen levels at day 2 post-infection in NL4-3-infected cells (A) and at day 4 post-infection in BaL-infected cells (B) in untreated, CD- and cholesterol-replenished (CD+Cho) cells. Data shown are the mean ± SD of triplicate points (n = 4). *Statistically significant difference, p <0.001 (two-tailed t-test). (C) NL4-3-induced syncytia in a representative field of control, CD- and CD+Cho-infected MT-2-CCR5 cells (100×). (D) Representative p24 production kinetics in untreated (control), CD-treated and cholesterol-replenished (CD+Cho) NL4-3-infected cells. (E) NL-4-3-infected MT-2-CCR5 cells (day 2 post-infection) were CD and cholesterol treated. After extensive washing, 106 cells were plated in complete RPMI and p24 production measured in ELISA after 24 h (black bars) and 48 h (white bars). (F) Untreated or CD-treated MT-2 cells were infected with a replication-defective NL4-3 variant, or with a VSV-G- or MLV-env-pseudotyped NL4-3 virus. Two days later, infected cells were determined. The percentage of CD-induced inhibition for each viral type is shown.

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Fig. 4. Cholesterol depletion inhibits env-mediated fusion. (A) Untreated (control) or CD-treated MT-2 cells were incubated alone (gray area) or with gp120 (dotted line) and binding monitored in flow cytometry. (B) Untreated or CD-treated MT-2 or HEK-293, as a CD4– cell, were incubated with NL4-3 and, after removal of unbound virus, cell lysates prepared. Total lysates were resolved by SDS–PAGE and sequentially blotted with anti-gp120 and anti-tubulin antibodies, as indicated. (C) Untreated or CD-treated MT-2 cells were exposed to NL4-3 as in Figure 3, and after 6 h culture, total DNA was amplified by PCR with primers specific for HIV gag. Either 100 or 10 ng of total DNA from the PCR products were hybridized in solution with an internal 32P-labeled gag probe, resolved by SDS–PAGE and autoradiographed. Serial dilutions of proviral 8E5 DNA were run in parallel for standardization. HLA-DQα amplification is shown as a loading control. (D) Untreated, CD-treated and cholesterol-replenished (CD+Cho) HeLa-CD4 cells were mixed with BSC40-env cells and cell fusion events measured. *Statistically significant difference, p <0.001 (two-tailed t-test).

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