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-␤-cyclodextrin (M␤CD) 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-1␣-and macrophage inflammatory protein 1␤-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 M␤CD 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.

Ceramide, a target for antiretroviral therapy

“…CD4 appears to be localized in cholesterol and glycosphingolipid-enriched membrane microdomains (rafts) (13). Experimental results suggest that integrity of lipid rafts is important for HIV-1 entry, and destabilizing rafts by extraction of cholesterol inhibits HIV-1 entry (14)(15)(16)(17)(18)(19). However, studies with CD4 mutants that do not partition into rafts yielded conflicting results, which indicate that localization of CD4 in rafts is essential (20), or nonessential (21,22), for HIV-1 entry.…”

Evidence That HIV Budding in Primary Macrophages Occurs through the Exosome Release Pathway

“…Several other viruses also require the integrity of lipid rafts on target cells for infection, including filoviruses (9) and measles virus (10). Recent studies showing that lipid rafts are present on HIV particles and that depletion of viral membrane cholesterol blocks HIV infection further highlight the critical role of lipid rafts in the biology of this virus (11)(12)(13)(14).…”

CD4 and CCR5 Constitutively Interact at the Plasma Membrane of Living Cells

“…In support of this view, high-resolution electron microscopy-based approaches have demonstrated that CCR5, CXCR4, and CD4 form homogeneous microclusters on cell surface microvilli in primary macrophages and T cells (6). The requirement of cholesterol for chemokine receptor functions and HIV entry (7)(8)(9) led to the hypothesis that cholesterol-and sphingolipid-enriched raft membrane domains represent privileged sites in which receptors localize. Nonetheless, the observations that coreceptors barely associate with rafts (8,10,11) and that CD4 mutants localizing to non-raft domains are fully competent for HIV entry (8,12) challenged this view.…”

“…It is likely that cholesterol outside rafts modulates the CD4 to CCR5 interactions we demonstrate in this work. Indeed, it was recently reported that cholesterol controls lateral distribution of CD4 and co-receptors at the plasma membranes of host cells, which in turn may be of importance for HIV entry (9). Current evidence shows that biological membranes are composed of a great diversity of domains (58,59), so that the composition and features of those where CD4 and co-receptors interact and segregate for HIV entry are far from being fully characterized.…”