Synthesis of single- and double-chain fluorocarbon and hydrocarbon galactosyl amphiphiles and their anti-HIV-1 activity.

Galactosylceramide (GalCer) is an alternative receptor allowing HIV-1 entry into CD4(-)/GalCer(+) cells. This glycosphingolipid recognizes the V3 loop of HIV gp120, which plays a key role in the fusion of the HIV envelope and cellular membrane. To inhibit HIV uptake and infection, we designed and synthesized analogs of GalCer. These amphiphiles and bolaamphiphiles consist of single and double hydrocarbon and/or fluorocarbon chain beta-linked to galactose and galactosamine. They derive from serine (GalSer), cysteine (GalCys), and ethanolamine (GalAE). The anti-HIV activity and cytotoxicity of these galactolipids were evaluated in vitro on CEM-SS (a CD4(+) cell line), HT-29, a CD4(-) cell line expressing high levels of GalCer receptor, and/or HT29 genetically modified to express CD4. GalSer and GalAE derivatives, tested in aqueous medium or as part of liposome preparation, showed moderate anti-HIV-1 activities (IC50 in the 20-220 microM range), whereas none of the GalCys derivatives was found to be active. Moreover, only some of these anti-HIV active analogs inhibited the binding of [3H]suramin (a polysulfonyl compound which displays a high affinity for the V3 loop) to SPC3, a synthetic peptide which contains the conserved GPGRAF region of the V3 loop. Our results most likely indicate that the neutralization of the virion through masking of this conserved V3 loop region is not the only mechanism involved in the HIV-1 antiviral activity of our GalCer analogs.

Synthesis of glycolipid analogues that disrupt binding of HIV-1 gp120 to galactosylceramide.

HIV-1 has been shown to infect CD4 negative cells by the binding of HIV gp120 to the glycolipid galactosylceramide (1) (GalCer). Several analogues of 1 were prepared to investigate the specific orientation of 1 in the membrane bilayer that is involved in gp120 binding. Interestingly, N-stearyl-1-deoxynojirimycin (8) displayed potent and specific affinity for gp120 equal to that of 1, a finding that may shed light on the antiviral activity of N-butyl-1-deoxynojirimycin.

Role of glycosphingolipid microdomains in CD4-dependent HIV-1 fusion.

The fusion of HIV-1 with the plasma membrane of CD4+ cells is triggered by the interaction of HIV-1 surface envelope glycoprotein gp120 with the CD4 receptor, and requires coreceptors (CCR5 and CXCR4). Recent advances in the study of HIV-1 entry into CD4+ cells suggest that glycosphingolipids (GSL) may also participate in the fusion process. GSL are organized in functional microdomains which are associated with specific membrane proteins such as CD4. GSL-enriched microdomains were purified from human lymphocytes and reconstituted as a monomolecular film at the air-water interface of a Langmuir film balance. Surface pressure measurements allowed to characterize the sequential interaction of GSL with CD4 and with gp120. Using this approach, we identified globotriaosylceramide (Gb3) and ganglioside GM3 as the main lymphocyte GSL recognized by gp120. In both cases, the interaction was saturable and dramatically increased by CD4. We propose that GSL microdomains behave as moving platforms allowing the recruitment of HIV-1 coreceptors after the initial interaction between the viral particle and CD4. According to this model, the GSL microdomain may: i) stabilize the attachment of the virus with the cell surface through multiple low affinity interactions between the V3 domain of gp120 and the carbohydrate moiety of GSL, and ii) convey the virus to an appropriate coreceptor by moving freely in the outer leaflet of the plasma membrane. This model can be extrapolated to all envelope viruses (e.g. influenza virus) that use cell surface GSL of the host cells as receptors or coreceptors.

Glycosphingolipid (GSL) microdomains as attachment platforms for host pathogens and their toxins on intestinal epithelial cells: activation of signal transduction pathways and perturbations of intestinal absorption and secretion.

Glycosphingolipid (GSL)-enriched microdomains are used as cellular binding sites for various pathogens including viruses and bacteria. These attachment platforms are specifically associated with transducer molecules, so that the binding of host pathogens (or their toxins) to the cell surface may result in the activation of signal transduction pathways. In the intestinal epithelium, such pathogen-induced dysregulations of signal transduction can elicit a severe impairment of enterocytic functions. In this study, we demonstrate that the interaction of a bacterial toxin (cholera toxin) and a viral envelope glycoprotein (HIV-1 gp120) with the apical plasma membrane of intestinal cells is mediated by GSL-enriched microdomains that are associated with G regulatory proteins. These microbial proteins induce a GSL-dependent increase of intestinal fluid secretion by two mechanisms: activation of chloride secretion and inhibition of Na+ -dependent glucose absorption. Taken together, these data support the view that GSL-enriched microdomains in the apical plasma membrane of enterocytes are involved in the regulation of intestinal functions.

Human erythrocyte glycosphingolipids as alternative cofactors for human immunodeficiency virus type 1 (HIV-1) entry: evidence for CD4-induced interactions between HIV-1 gp120 and reconstituted membrane microdomains of glycosphingolipids (Gb3 and GM3).

Glycosphingolipids from human erythrocytes mediate CD4-dependent fusion with cells expressing human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins. To identify the glycosphingolipid(s) which participates in the fusion process, we have analyzed the interaction of HIV-1 gp120 (X4 and R5X4 isolates) with reconstituted membrane microdomains of human erythrocyte glycosphingolipids. We identified globotriaosylceramide (Gb3) and ganglioside GM3 as the main glycosphingolipids recognized by gp120. In the presence of CD4, Gb3 interacted preferentially with the X4 gp120, whereas GM3 interacted exclusively with the R5X4 gp120. These data suggest that glycosphingolipid microdomains are required in CD4-dependent fusion and that Gb3 and/or GM3 may function as alternative entry cofactors for selected HIV-1 isolates.

HIV-1-induced perturbations of glycosphingolipid metabolism are cell-specific and can be detected at early stages of HIV-1 infection.

The metabolism of glycosphingolipids (GSL) has been investigated in peripheral blood mononuclear cells (PBMC) from 8 patients at an early stage of HIV-1 infection. Following metabolic labeling of these cells with [14C]galactose, the GSL were purified and the radioactivity incorporated into each individual GSL quantitated by phosphoimaging. Compared with PBMC from seronegative donors, the GSL metabolism in PBMC from HIV-1-infected individuals was characterized by an increased synthesis of two GSL: the B-lymphocyte differentiation antigen globotriaosylceramide (Gb3, also referred to as CD77), and the monosialoganglioside GM3, a marker of T-lymphocytes and macrophages. The accumulation of Gb3 and GM3 in PBMC from HIV-1-infected patients was associated with the appearance of anti-Gb3 and anti-GM3 antibodies. Because these GSL are involved in the control of cell proliferation and signal transduction, such anti-GSL autoantibodies may contribute to the immune suppression during the course of HIV-1 infection. Studies on purified cell populations showed that GM3 accumulation occurred preferentially in HIV-1-infected monocytes/macrophages, whereas the synthesis glucosylceramide, the common precursor of complex GSL, was enhanced in both macrophages and CD4+ lymphocytes. Taken together, our data suggest that the dysregulation of GSL metabolism is an early event of HIV-1 pathogenesis that can induce important effects on immune cells homeostasis.

Sulfatide inhibits HIV-1 entry into CD4-/CXCR4+ cells.

Sulfatide (3'sulfogalactosylceramide) is the natural sulfated derivative of galactosylceramide (GalCer), a glycosphingolipid receptor allowing HIV-1 infection of CD4-negative cells from neural and intestinal tissues. The incorporation of exogenous sulfatide into the plasma membrane of HT-29 (a CD4-/GalCer+/CXCR4+ human intestinal cell line) or RD (CD4-/GalCer-/ CXCR4+ human rhabdomyosarcoma) resulted in a dose-dependent inhibition of HIV-1 infection. Experiments with luciferase reporter viruses pseudotyped with HIV-1 or amphotropic murine leukemia virus envelopes demonstrated that sulfatide acts at the level of viral entry. Paradoxically, the transfer of sulfatide in the plasma membrane of various CD4- cells resulted in increased binding of HIV-1. Surface pressure measurements were conducted to study the interaction of gp120 with glycosphingolipid monolayers. The data showed that gp120 could penetrate into a monomolecular film of GalCer, confirming the role of this glycosphingolipid as a functional receptor for HIV-1. In contrast, the insertion of gp120 into a monolayer of sulfatide was very limited. Moreover, the incorporation of sulfatide in a monomolecular film of GalCer specifically inhibited the penetration of gp120. In conclusion, these data show that sulfatide mediates gp120 binding but, in marked contrast with GalCer, is not able to initiate the fusion event.

Sequential interaction of CD4 and HIV-1 gp120 with a reconstituted membrane patch of ganglioside GM3: implications for the role of glycolipids as potential HIV-1 fusion cofactors.

The fusion of HIV-1 with CD4+ cells involves, in addition to CD4, specific cell surface molecules acting as fusion cofactors. Recently, we reported that the V3 loop of HIV-1 gp120 binds to GM3, a ganglioside abundantly expressed on CD4+ lymphocytes and macrophages. In the present study, we show that CD4 interacts with a reconstituted patch of GM3 by measuring the surface pressure with a Langmuir film balance. A biphasic increase in surface pressure is observed after the sequential addition of CD4 and gp120 under the GM3 monolayer, indicating the formation of the trimolecular complex GM3-CD4-gp120. Neutralization of gp120 with an anti-V3 antibody inhibits the secondary interaction with GM3, suggesting that the CD4-induced conformational change in gp120 allows the V3 loop to interact with GM3. In conclusion, this study supports the concept that glycolipids can function as HIV-1 fusion cofactors.

Specific interaction of HIV-1 and HIV-2 surface envelope glycoproteins with monolayers of galactosylceramide and ganglioside GM3.

Cellular glycosphingolipids mediate the fusion between some viruses and the plasma membrane of target cells. In the present study, we have analyzed the interaction of human immunodeficiency virus (HIV)-1 and HIV-2 surface envelope glycoproteins from distinct viral isolates with monolayers of various glycosphingolipids at the air-water interface. The penetration of the viral glycoproteins into glycosphingolipid monolayers was detected as an increase in the surface pressure. We found that HIV-1 recombinant gp120 (IIIB isolate) could penetrate into a monomolecular film of alpha-hydroxylated galactosylceramide (GalCer-HFA), while ceramides, GluCer, and nonhydroxylated GalCer were totally inactive. The glycoproteins isolated from HIV-1 isolates LAI and NDK and from HIV-2(ROD) could also interact with a GalCer-HFA monolayer, whereas gp120 from HIV-1(SEN) and HIV-1(89.6) did not react. These data correlated with the ability of the corresponding viruses to gain entry into the CD4(-)/GalCer+ cell line HT-29, demonstrating the determinant role of GalCer-HFA in this CD4-independent pathway of HIV-1 and HIV-2 infection. In contrast, all HIV-1 and HIV-2 glycoproteins tested were found to interact with a monolayer of GM3, a ganglioside abundantly expressed in the plasma membrane of CD4(+) lymphocytes and macrophages. A V3 loop-derived synthetic peptide inhibitor of HIV-1 and HIV-2 infection in both CD4(-) and CD4(+) cells could penetrate into various glycosphingolipid monolayers, including GalCer-HFA and GM3. Taken together, these data suggest that the adsorption of human immunodeficiency viruses to the surface of target cells involves an interaction between the V3 domain of the surface envelope glycoprotein and specific glycosphingolipids, i.e. GalCer-HFA for CD4(-) cells and GM3 for CD4(+) cells.

Glycolipids as potential binding sites for HIV: topology in the sperm plasma membrane in relation to the regulation of membrane fusion.

Although human sperm cells can bind human immunodeficiency virus (HIV-1), they lack CD4, galactoceramides (GalCer) and sulfogalactoceramides (SGalCer) as gp120 receptors. However, sperm specific glycolipids (sulfogalactosylalkylacylglycerol (SGalAAG) and galactosylalkylacylglycerol (GalAAG)) are structurally closely related to SGalCer and GalCer as predicted by computer simulated molecular modelling. SGalAAG and GalAAG are exclusively localized in the outer leaflet of the human sperm plasma membrane, and therefore we tested whether they could serve as alternative receptors for the gp120. Purified SGalAAG and GalAAG had similar affinities to recombinant gp120 as the hydroxy fatty acid (HFA) SGalCer and HFA-GalCer respectively. However, nonhydroxy fatty acid forms of (S)GalCer, galactosyldiacylglycerol and the deacylated (sulfo)galactosyllipids did not recognize recombinant gp120. Data obtained by surface pressure experiments revealed that the lipid monolayers that contained HFA-GalCer or GalAAG resulted in a similar significant penetration of recombinant gp120 in the monolayer. The penetration was a factor of two lower in monolayers with HFA-SGalCer or SGalAAG. The binding of recombinant gp120 to human sperm cells colocalized with GalAAG and could be blocked with monoclonal antibodies against galactolipids. The possible relevance of gp120 binding to glycolipids for HIV entry in sperm cells is discussed.

Co-expression of CXCR4/fusin and galactosylceramide in the human intestinal epithelial cell line HT-29.

OBJECTIVE: To detect the expression CXCR4/fusin in human intestinal epithelial cells and to assess its potential role in the pathway of HIV-1 infection mediated by the alternative gp120 receptor galactosylceramide (GalCer). METHODS: GalCer+ (HT-29, HT-29/CD4+) and GalCer- (Caco-2/Cl2, Cl14 and Cl14/CD4+) human intestinal cell lines were analysed for CXCR4/fusin expression using the monoclonal antibody (MAb) 12G5. This MAb was then evaluated for its ability to inhibit HIV-1 infection in permissive cells. HIV-1 infection was measured by detection of p24 antigen, polymerase chain reaction amplification, and cocultivation with CD4+ cells. RESULTS: CXCR4/fusin was detected on the surface of HT-29 and HT-29/CD4+, but not on Caco-2/Cl2, Cl14 and Cl14/CD4+ cells. Ninety per cent of CXCR4/fusin+ HT-29 and HT-29/CD4+ cells co-expressed GalCer. Infection of HT-29 cells by laboratory isolates of HIV-1 was inhibited by both anti-GalCer and anti-CXCR4/fusin MAbs. Expression of CD4 rendered HT-29 cells sensitive to HIV-1(89.6), a macrophage-tropic isolate that does not recognize GalCer. The 12G5 MAb blocked HIV-1 infection of HT-29/CD4+ cells. In contrast, the expression of HIV-1 receptors, i.e., CD4 GalCer or both, into CXCR4/fusin-negative intestinal cells did not confer sensitivity to HIV-1 infection. The resulting receptor-positive cell lines could, however, bind HIV-1, whereas the original cell lines could not. CONCLUSION: HIV-1 entry into human intestinal cells involves both GalCer and CXCR4/fusin. HIV-1 isolates such as 89.6 that are able to use CXCR4/fusin as coreceptor, but do not bind to GalCer, do not infect these cells. These data raise the possibility that CXCR4/fusin may function as a coreceptor for HIV-1 entry into CD4-/GalCer+ intestinal epithelial cells.

Synthetic soluble analogs of galactosylceramide (GalCer) bind to the V3 domain of HIV-1 gp120 and inhibit HIV-1-induced fusion and entry.

Galactosylceramide (GalCer) is an alternative receptor allowing human immunodeficiency virus (HIV)-1 entry into CD4-negative cells of neural and colonic origin. Several lines of evidence suggest that this glycosphingolipid recognizes the V3 region of HIV-1 surface envelope glycoprotein gp120. Since the V3 loop plays a key role in the fusion process driven by HIV-1, we decided to synthesize soluble analogs of GalCer with the aim to develop a new class of anti-HIV-1 agents that could neutralize HIV-1 infection through masking of the V3 loop. We describe a short route, in three steps, for the synthesis of soluble analogs of GalCer, using unprotected lactose as the starting sugar. The analogs were prescreened in an assay based on the interaction between a V3 loop-derived synthetic peptide and [3H]suramin, a polysulfonyl compound displaying high affinity for the V3 loop. One of the soluble analogs, i.e. CA52(n15), strongly inhibited the binding of [3H]suramin to the V3 peptide, with an IC50 of 1.2 microM. This molecule was also able to inhibit [3H]suramin binding to recombinant gp120 with similar activity. Using a competition enzyme-linked immunosorbent assay with highly specific anti-gp120 monoclonal antibodies, the region recognized by CA52(n15) could be mapped to amino acids 318-323, which corresponds to the highly conserved consensus motif GPGRAF. Interestingly, the region recognized by suramin, i.e. IQRGP-R-F, was partially overlapping this motif. CA52(n15) was able to inhibit HIV-1-induced cell fusion as well as HIV-1 entry into both CD4(+) and CD4(-)/GalCer+ cells. A structure-activity relationship study showed that: (i) the antiviral activity of soluble analogs of GalCer correlates with V3 loop binding, and (ii) the hydrophobic moiety of the molecule plays an important role in this activity. Taken together, these data show that synthetic analogs of GalCer can inhibit HIV-1 entry into both CD4(-) and CD4(+) cells through masking of the V3 loop.

SPC3, a V3 loop-derived synthetic peptide inhibitor of HIV-1 infection, binds to cell surface glycosphingolipids.

Synthetic multibranched peptides derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120 inhibit HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms: competitive inhibition of HIV-1 binding to CD4-/GalCer+ colon cells and postbinding inhibition of HIV-1 fusion with CD4+ lymphocytes. In the present study, we have characterized the cellular binding sites for the V3 peptide SPC3, which possesses eight V3 consensus motifs GPGRAF radially branched on a neutral polyLys core matrix. These binding sites are glycosphingolipids that share a common structural determinant, i.e., a terminal galactose residue with a free hydroxyl group in position 4: GalCer/sulfatide on CD4-/GalCer+ colon cells; LacCer and its sialosyl derivatives GM3 and GD3 on CD4+ human lymphocytes. These data suggest that the V3 peptide binds to the GalCer/sulfatide receptor for HIV-1 gp120 on HT-29 cells and thus acts as a competitive inhibitor of virus binding to these CD4- cells, in full agreement with previously published virological data. In contrast, SPC3 does not bind to the CD4 receptor, in agreement with the data showing that the peptide inhibits HIV-1 infection of CD4+ cells by acting at a postattachment step. The binding of SPC3 to LacCer, GM3, and GD3, expressed by CD4+ lymphocytes, suggests a role for these glycosphingolipids in the fusion process between the viral envelope and the plasma membrane of CD4+ cells. Since the multivalent peptide can theoretically bind to several of these glycosphingolipids, we hypothesize that the resulting cross-linking of membrane components may affect the fluidity of the plasma membrane and/or membrane curvature, altering the virus-cell fusion mechanism.