Glycosphingolipids Promote Entry of a Broad Range of Human Immunodeficiency Virus Type 1 Isolates into Cell Lines Expressing CD4, CXCR4, and/or CCR5

Abstract

Treatment of human osteosarcoma cells, expressing CD4 and various chemokine receptors, with the glucosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), blocked target membrane glycosphingolipid (GSL) biosynthesis and reduced the susceptibility of cells to infection and fusion mediated by envelope glycoproteins from a variety of human immunodeficiency virus type 1 (HIV-1) isolates that utilize CXCR4 and/or CCR5. PPMP treatment of the cell lines did not significantly change the cell surface expression of CD4, CXCR4, and/or CCR5, nor did it alter the chemokine receptor association with CD4. PPMP-treated cells exhibited no changes in chemokine-induced Ca 2؉ mobilization and chemotaxis. However, massive envelope glycoprotein conformational changes triggered by CD4 and the appropriate chemokine receptor on the target membrane were inhibited when the target cells were treated with PPMP. Addition of various purified GSLs to PPMP-treated target cells showed that for all isolates tested, globotriaosylceramide (Gb3) was the most potent GSL in restoring the fusion susceptibility of target cells with cells expressing HIV-1 envelope glycoproteins; addition of the monosialoganglioside GM3 yielded a slight enhancement of fusion susceptibility. Our data are consistent with the notion that a limited number of specific GSL species serve as crucial elements in organizing gp120-gp41, CD4, and an appropriate chemokine receptor into a membrane fusion complex.Human immunodeficiency virus type 1 (HIV-1) infects susceptible cells by fusion of the viral membrane with the cell plasma membrane. This process is mediated by the interactions of the HIV-1 envelope glycoprotein gp120-gp41 (11,45,46) with CD4 on the host cell surface (26) and requires additional coreceptors such as CXCR4 (X4) and CCR5 (R5) (4,5,14,28), which determine the tropism of different HIV-1 isolates. Several viral envelope glycoprotein oligomers then assemble into a viral fusion machine (18, 23), forming a molecular scaffold that brings the viral and target cell membranes into close apposition and allow the actual fusion event (29). Previously reported work suggests that glycosphingolipids (GSLs) may be involved in the assembly and functioning of the HIV-1 fusion machine. This is based on the demonstration that inhibition of target cell GSL biosynthesis affects HIV-1 envelope glycoprotein-mediated cell-cell fusion (37) and that fusion activity can be recovered following addition of human erythrocyte membrane components (15, 38) or purified GSLs to the impaired cells (35). Moreover, studies using reconstituted monolayers of purified GSLs at the air-water interface provide evidence for CD4-induced interactions between HIV-1 gp120 and the GSLs globotriaosylceramide (Gb3) and the monosialoganglioside GM3 (21). These observations have led to the hypothesis that plasma membrane GSL microdomains are preferential sites for assembly of the HIV-1 fusion machine (21,36).In this study, we have examined the effect of treating a variety of cell ...

CD4 Receptor Localized to Non-raft Membrane Microdomains Supports HIV-1 Entry

“…Experimental results suggest that integrity of lipid rafts is important for virus entry, and destabilizing rafts by either extraction of cholesterol or inhibition of glycosphingolipid synthesis inhibits virus entry (13,17,42). However, extraction of cholesterol with ␤-cyclodextrin suffers from various drawbacks, thereby raising questions about specificity of the observed inhibition of virus entry.…”

“…Although conformation and function of these coreceptors depend on membrane cholesterol (13)(14)(15) and the interaction with glycosphingolipids (16) during virus entry (17), HIV-1 coreceptors may localize to microdomains different from that occupied by CD4 (18,19). Thus, activation of the entry process through engagement of CD4 and one of the coreceptors, would require a mechanism allowing for the apposition of the entry receptors in the same membrane environment.…”

Chemokine receptor internalization and intracellular trafficking

“…The observation that CCR5 and CXCR4 are constitutively associated with other raft proteins, such as CD4, provides indirect evidence for their raft localization [40]. Moreover, inhibition of glycosphingolipid synthesis reduces the CCR5 levels in cell membranes, suggesting association of CCR5 with rafts for proper receptor transport [41]. Although CXCR1 and CXCR2 are soluble in Triton X-100 and co-patched with non-raft rather than raft markers, a fraction of CXCR1 and CXCR2 is associated with alternative cholesterol-based rafts [42].…”

Identification of a Common Sphingolipid-binding Domain in Alzheimer, Prion, and HIV-1 Proteins

“…the ceramide trihexoside Gb3, the monosialoganglioside GM3, and sphingomyelin (6 -10). Raft glycosphingolipids mediate lateral assemblies of the HIV-1 fusion complex and stimulate the conformational changes in HIV-1 envelope glycoproteins required for initiating the fusion process (9,(11)(12)(13). Moreover, a major conformational transition of the Alzheimer ␤-amyloid peptide is observed upon binding of the 1-40 ␤-amyloid peptide to ganglioside GM1-containing membranes (14).…”