Objective: To determine the effect of a gp120 binding, non-cytotoxic soluble analogue of the glycosphingolipid (GSL), globotriaosyl ceramide (Gb3) on HIV infection in vitro.
Design: HIV-1IIIB (X4 virus) infection in Jurkat and phytohaemagglutinin (PHA)/interleukin-2 (IL2) activated, peripheral blood mononuclear cells (PBMC), and HIV-1Ba-L (R5 virus) infection of PHA activated PBMC in vitro were assessed. We monitored cell surface markers, cell viability, and viral/host cell morphology to eliminate pleiotropic effects. Viral-host cell fusion was measured to further address any inhibitory mechanism.
Methods: HIV infection was monitored by p24gag ELISA. CD4, CCR5, CXCR4 and apoptosis were determined by fluorescent antibody cell sorting. A model fusion system comprising a cell line transfected with either CD4 and CXCR4 or CCR5, cocultured with a cell line expressing gp120 from either X4-, R5-tropic HIV-1 or HIV-2 virions, was used. PHA/IL2 activated PBMC GSL synthesis was monitored by metabolic radiolabelling.
Results: AdamantylGb3 blocked X4 and R5 virus infection with a 50% inhibitory concentration of approximately 150 μM. A reverse transcriptase and a protease-resistant X4 HIV-1 strain retained adamantylGb3 sensitivity. AdamantylGb3 had minimal effect on cell viability. Treated Jurkat cells showed a small increase in CCR5/CXCR4 expression and a slight, transient CD4 down-regulation, which was probably not related to the mechanism of inhibition. Electron microscopy showed normal viral and host cell morphology following adamantylGb3 treatment, and viral entry was blocked. AdamantylGb3 was able to prevent virus-host cell fusion irrespective of HIV strain or chemokine receptor preference.
Conclusions: These results suggest that adamantylGb3 may provide a new basis for blocking HIV infections, irrespective of HIV envelope/chemokine co-receptor preference or resistance to other therapeutics.
From the aDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Canada
bResearch and Development of Canadian Blood Services, Toronto, Ontario M5G 2M1 Canada
cDepartment of Medicine, University of Toronto, Canada
dDivision of Cell and Molecular Biology, Toronto General Research Institute of the University Health Network, Canada
eResearch Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8 Canada
fInstitut Méditerranéen de Recherche en Nutrition, Faculté des Sciences, St-Jérôme, Marseille, France
gCenter for Cancer Research Nanobiology Program, National Cancer Institute Frederick, Maryland, USA
hDepartment of Biochemistry, University of Toronto, Canada.
Received 2 August, 2005
Revised 7 September, 2005
Accepted 3 October, 2005
Correspondence to D.R. Branch, Toronto General Research Institute, 67 College St., Toronto, Ontario M5G 2M1 Canada or C.A. Lingwood, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada. Tel: +1 416 813 5998; fax: +1 416 813 5993; e-mail: firstname.lastname@example.org
HIV targeting of CD4 and chemokine co-receptor expressing lymphoid and monocytic cells has long been appreciated as the major mechanism of HIV–host cell interaction [1–3]. In addition to CD4 and the chemokine co-receptors, gp120 shows an affinity in vitro for several glycosphingolipids (GSL) [4–6]. These include galactosyl ceramide, sulfogalactosyl ceramide and GM3 ganglioside. This binding is mediated by the V3 loop on gp120  via a sphingolipid recognition motif , which facilitates a post-CD4 binding event to allow the host cell entry of diverse HIV strains . It has become clear that, as with many microbial infections, GSL-enriched lipid microdomains on the host cell surface are central to HIV [9,10] and inhibition of GSL synthesis protects cells from infection . GSL may facilitate simultaneous CD4/co-receptor binding since overexpression of transfected CD4 and co-receptor can reduce the GSL requirement . The broad GSL binding selectivity of gp120 did not promote the idea of a specific role for GSL recognition in HIV infection, despite the fact that analogues of galactosyl ceramide could be effective inhibitors of infection in vitro . However, the demonstration that the GSL, globotriaosyl ceramide (Gb3) was selectively involved in the mechanism of HIV–host cell fusion  has led to a more concerted investigation of the potential role this GSL plays in the infection cycle.
Gb3 is the receptor for the Escherichia coli elaborated verotoxin (VT),  which is involved in the etiology of hemolytic uremic syndrome, primarily a glomerular vasculopathy of young children . The binding of VT to Gb3 is dependent not only on the sugar, but on the aglycone . Similarly, the aglycone influences gp120/carbohydrate binding [9,17]. We have designed a novel soluble analogue of Gb3 which satisfies the aglycone requirement in an aqueous milieu . In this GSL mimic, the fatty acid was replaced with a globular adamantane frame, the rigidity of which is necessary for its receptor mimic function . We have shown adamantylGb3 to be a highly effective ligand for HIV gp120 as monitored by insertion into water/air interface adamantylGb3 monolayers . Insertion into Gb3 monolayers showed a 1–2 h lag period followed by slow sigmoidal insertion kinetics, whereas gp120 insertion into adamantylGb3 monolayers was instantaneous, exponential and complete within 10 min. Thus, adamantylGb3 is a superior receptor for gp120. We have now investigated the effect of adamantylGb3 on HIV infection in culture.
Cells, vaccinia constructs and other reagents
All cells were cultured in complete RPMI1640 medium (Invitrogen Canada, Burlington, Ontario) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 10 μM gentamicin antibiotics. The human T-cell line, Jurkat FHCRC (Jurkat C), was a gift from G. Mills (MD Anderson Cancer Center, Houston, Texas, USA) and Jurkat clone E6-1 was obtained from American Type Culture Collection (ATCC, Rockville, Maryland, USA). MT-4 cells and NIH 3T3 fibroblasts stably expressing human CD4 and either human CXCR4 or CCR5 were from the NIH AIDS Research and Reference Reagent Program (Rockville, Maryland, USA). Coreceptor-expressing NIH3T3 cells were grown in DMEM supplemented with 10% FBS, antibiotics and puromycin (3 μg/ml). HeLa cells (ATCC) were used for transient expression of HIV-1 and HIV-2 envelope glycoproteins by infection with recombinant vaccinia virus vectors, vSC50, vPE16 or vCB43. vSC50 (encoding the full length env gene from HIV-2SBL/ISY)  was obtained through the NIH AIDS Research and Reference Program. vPE16 (encoding the full length env gene from HIV-1IIIB clone BH8)  and vCB43 (encoding the full length env gene from HIV-1Ba-L)  were gifts from P. Earl, C.C. Broder and B. Moss. Peripheral blood mononuclear cells (PBMC) were prepared from whole blood obtained from healthy volunteers after informed consent using Ficoll-Paque (Amersham Pharmacia Biotech Inc., Baie d'Urfe, Quebec, Canada) density gradients . Activated PBMC were produced by stimulation for 2–3 days in 5 μg/ml phytohemagglutinin (PHA, Sigma-Aldrich Chemical, Oakville, Ontario) followed by 2 days' stimulation with interleukin-2 (IL-2; Invitrogen, 100 U/ml) resulting in 90–95% proliferating T cell blasts . For experiments using R5 viruses, only PHA for 2 days was used to activate PBMC and minimize T-cell proliferation. Fluorescent dyes, CMFDA and CMTMR were from Molecular Probes, Inc. (Eugene, Oregon, USA).
2-(1-Adamantanacetamido)-3-hydroxyl-octadec-4-enyl (αD-galactopyranosyl)-(1-4)-β-galactopyranosyl)-(1-4)-β-D-glucopyranoside (AdamantylGb3)
Gb3 was purified from human kidney, deacylated and coupled to α adamantane .
Viruses and in vitro infections
X4 HIV-1IIIB, and R5 HIV-1Ba-L, HIV-1RT-MDR1 and HIV-1 (saquinavir resistant), were from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HTLVIIIB (HIV-1IIIB) R. Gallo, HIV-1Ba-L S. Gartner, M. Popovic and R. Gallo, HIV-1RT-MDR1 B. Larder, HIV-1 (saquinavir resistant) from N. Roberts and P. Tomlinson. Viral stocks were grown in Jurkat C (HIV-1IIIB) or PBMC (HIV-1RT-MDR1, HIV-1 (sasquinavir resistant), and R5 HIV-1Ba-L) and multiplicity of infection (m.o.i) was determined using MT-4 cells for HIV-1IIIB  or calculated based on total p24gag. Infections were carried out in a Level 3 containment facility (University of Toronto) as described  with minor modification. Briefly, HIV-1 was incubated with adamantylGb3 over the concentration range 25–400 μM or with adamantamine control (400 μM) for 1 h at 37°C prior to infection. Then 5 × 105 Jurkat or activated PBMC were infected for 1 h at 37°C with untreated, adamantamine treated or adamantylGb3 treated HIV-1IIIB (Jurkat and PBMC) or HIV-1Ba-L (PBMC). For infections using HIV-1Ba-L, PBMC were treated with 2 μg/ml polybrene (Sigma-Aldrich Chemical) for 30 min at 37°C prior to infection. For infections using HIV-1RT-MDR1 and HIV-1 (saquinavir resistant) strains, 2.5 × 105 PBMC were infected with 400 μM adamantylGb3 or adamantamine treated virus in MultiscreenHTS 96-well filtration plates (Durapore-DV 0.65 μm, Millipore, Etobicoke, Ontario, Canada) followed by filtering, according to manufacturer's instructions. After infection, the cells were washed extensively with phosphate buffered saline (PBS) without MgCl2/CaCl2, and cultured in complete RPMI1640 medium. Aliquots of culture supernatant were taken 2 h after initial infection and each day thereafter for measurement of p24gag antigen production by ELISA (Coulter, Miami, Florida, USA).
To determine the effects of adamantylGb3 on viability and/or cell surface receptor expression, 5 × 105 Jurkat cells or activated PBMC were incubated with 200–400 μM adamantylGb3 for 1–4 h at 37°C. For surface receptor expression after 1 h of treatment, Jurkat cells were incubated for 20 min at 4°C with 10% human AB serum (volunteer healthy donor after informed consent) in PBS. Cells were re-suspended in 100 μl FACS Buffer (PBS, 2% FBS, 0.1% sodium azide, 5 mM EDTA) and 12.5 μg/ml monoclonal mouse anti-CCR5 (clone 2D7, NIH AIDS Research and Reference Reagent Program) was added. PBMC were incubated for 30 min at 4°C, followed by one wash with FACS buffer. Then, 1 μl fluoresceine isothiocyanate (FITC)-labelled goat anti-mouse (Sigma-Aldrich) was added and the cells were incubated for 30 min at 4°C in the dark, followed by one wash with FACS buffer. Jurkat cell aliquots were incubated with 20 μl of 10% mouse serum in FACS buffer for 10 min in the dark, prior to addition of 5 μl mouse anti-CD4-PerCP Cy5.5 (BD Biosciences) and/or 5 μl of mouse anti-CXCR4-PE (Serotec). Cells were incubated for 30 min at 4°C in the dark, followed by one wash with FACS buffer. For viability assays, treated cells were cultured at 37°C for 24 h in complete RPMI1640 media. Cells were washed once with PBS, and the cell pellet prepared using TACSTM AnnexinV-FITC apoptosis detection kit (R&D Systems, Minneapolis, Minnesota, USA). Briefly, cells were incubated with 0.5 μl Annexin V-FITC and 10 μl propidium iodide (PI) in 1 × binding buffer (total volume 100 μl) for 15 min at room temperature in the dark, followed by the addition of 500 μl 1× binding buffer. Data was collected with the Becton Dickenson FACSCalibur cell cytometer and analyses carried out using Cell Quest software.
Transmission electron microscopy
To visualize viral entry and fusion, X4 HIV-1IIIB was incubated for 1 h at 37°C with 150 μM (and 400 μM, data not shown) adamantylGb3 or adamantamine. Following incubation, 5 × 105 Jurkat E6 were incubated with pre-treated virus for 20 min at 37°C. Cells were centrifuged; pellets were resuspended in fixative (4% paraformaldehyde, 1% formaldehyde) and stored at 4°C prior to embedding and sectioning. Sections of 5 μm were cut, fixed postembedding with 1% osmium tetroxide and examined using a JEM1230 transmission electron microscope (JEOL, Peabody Massachusettes, USA).
To examine viral fusion in vitro for different gp120 tropisms, we used a fluorescent dye transfer assay . HIV-1 and HIV-2 envelope glycoproteins were transiently expressed on the surface of HeLa cells by infection with the recombinant vaccinia viruses, vPE16 (X4, HIV-1IIIB), vCB43 (R5, HIV-1Ba-L), or vSC50 (HIV-2SBL/ISY). NIH3T3CD4CXCR4 and NIH3T3CD4CCR5 cells were used as targets. Prior to fusion assay, envelope expressing HeLa cells, were labelled with the cell-tracking dye CMFDA (10 μM, excitation/emission (Ex/Em) 492/517 nm) and target NIH3T3 cell lines were labelled with CMTMR (10 μM, Ex/Em 541/565 nm) according to the manufacturer's instructions. Fluorescently labelled cells were resuspended in culture medium at 2 × 106 cells/ml. Duplicate samples of envelope-expressing cells (25 μl per well) were placed into a 96-well microtiter plate at a cell density of 5 × 104/well) and incubated with adamantylGb3 for 20–30 min. at room temperature. Controls were incubated in medium in the absence of adamantlyGb3. An equal number of NIH3T3CD4CXCR4 cells were added to HIV-1IIIB or HIV-2SBL/ISY-Env-expressing HeLa cells. NIH3T3CD4CCR5 cells were added to HIV-1Ba-L Env-expressing HeLa cells. Co-culture was continued at 37°C for 90 min. Images were collected using 20× lens (0.4NA objective) in an Olympus IX70 microscope. Positive fusion in multinucleated cells (syncytium) was confirmed by colocalization of CMFDA and CMTMR dyes. Percent fusion was determined by image analysis as described .
Metabolic labelling of PBMC GSL
PBMCs were activated with PHA and IL-2 as above. 14C-galactose (Amersham Biosciences) was added (2 μCi/mL) for a further 18 h at 37°C. Non-activated PBMCs were similarly labelled, 24 h after their isolation from blood. GSLs were extracted with chloroform/methanol (2: 1, v/v), partitioned against water and lower phase lipids saponified and desalted . An aliquot of the GSL fraction from activated PBMCs was digested with αgalactosidase overnight at 37°C . Equivalent aliquots were separated by TLC (C: M:W, 65: 25: 4) and the labelled species detected by autoradiography. Verotoxin1-TLC overlay was performed on an equivalent TLC plate to bind and selectively detect Gb3 .
AdamantylGb3 inhibits HIV-1 infection of Jurkat T cells
We first assessed the effect of adamantylGb3 on the infection of Jurkat T cells by HIV-1IIIB. Using an m.o.i. of 0.1 to permit reliable evaluation of infection via p24 expression after 3 days, we found (Fig. 1a) that pre-incubation of the virus with increasing doses of adamantylGb3 resulted in a substantial inhibition of Jurkat cell infection in vitro, where infection was increasingly inhibited over the dose range. Infection was reduced to background values at approximately 300 μM while 50% inhibition was achieved at approximately 150 μM adamantylGb3. Adamantamine, containing only the hydrophobic frame from which adamantylGb3 was prepared, showed no inhibition up to 400 μM. Treatment with adamantylGb3 for 1 h at 37°C, as compared to adamantamine, had no effect on Jurkat viability after 24 h as monitored by Annexin V-FITC/PI fluorescent antibody cell sorting (FACS) analysis measuring apoptosis (Fig. 1b). Nevertheless, Jurkat cells exposed to adamantylGb3 for 1 h showed a 14% reduction in cells expressing CD4 (Fig. 1c) although the cell surface CD4 mean fluorescence intensity (MFI) was not different (Fig. 1c). The percentage of cells expressing CXCR4 did not differ, although interestingly CXCR4 MFI appeared to be higher (Fig. 1c). These changes in CD4 and CXCR4 surface expression were transient, as they were not detected 24 or 72 h following treatment (data not shown). Surface CCR5, expressed at low levels in Jurkat cells was also slightly enhanced in adamantylGb3 treated cells (Fig. 1c). Pretreatment of Jurkat cells with 300 μM adamantylGb3 for 1 h at 37°C and washing out, showed no inhibition on subsequent HIV-1IIIB infection (not shown).
Electron microscopy reveals that adamantylGb3 has no deleterious effect on virions but prevents viral attachment to cells
Jurkat T cells exposed to adamantylGb3 treated virus, showed no abnormal host cell or viral morphology by electron microscopy (Fig. 2). In contrast to control HIV-1IIIB treated Jurkat cells, which showed cell attached virus and virions within the host cell cytoplasm (Fig. 2a), adamantylGb3 treated virus remained non-host cell attached and virions could not be found within the Jurkat cells (Fig. 2b and c). Viruses with an intact outer membrane and the triangular nucleocapsid core could be detected after adamantylGb3 treatment.
AdamantylGb3 inhibits HIV-1 infection of primary cells
To assess the potential for adamantylGb3 to inhibit HIV-1 infection under more physiological conditions, we examined the effects of adamantylGb3 on HIV-1 infection of primary lymphoid cells in vitro, as PBMC infection represents a closer approximation to natural in vivo infection. PBMCs were activated by treatment with both PHA and IL-2 to selectively induce the proliferation of T-lineage cells. Figure 3a shows that adamantylGb3 was effective at inhibiting HIV-1IIIB infection of PBMCs, with increasing inhibition over the dose range. This inhibitory effect was reproducible with complete inhibition attained at 300 μM adamantylGb3. Furthermore, treatment of drug resistant HIV-1RTMDR1 and sasquinavir resistant strains (2.5 ng p24gag/infection), showed inhibition comparable to HIV-1IIIB, at a 400 μM dose (Fig. 3c). The adamantamine control did not inhibit X4 HIV-1 infection in vitro. Infection with R5 HIV-1Ba-L (0.5 ng p24gag/infection) was robust but required pre-treatment with polybrene and a longer culture period to achieve similar p24 production as for HIV-1IIIB infections. Treatment of HIV-1Ba-L with increasing doses of adamantylGb3 showed similar inhibitory effects as seen for HIV-1IIIB; infection being completely inhibited by a 300 μM dose (Fig. 3b). The IC50 was approximately 150 μM adamantylGb3, similar to that for inhibition of T-tropic PBMC infection. Production of p24gag was monitored between 4 and 11 days post-infection, showing inhibition was maintained even after 11 days in culture (Fig. 3d and data not shown). The adamantamine control had no effect on PBMC HIV-1Ba-L infection up to 400 μM. Effects on PHA or PHA/IL-2 activated PBMC viability following adamantylGb3 treatment was minimal in comparison to the adamantamine control as shown by Annexin V-FITC/PI FACS analysis (Fig. 3e).
AdamantylGb3 inhibits fusion of both HIV-1 and HIV-2
To further define the inhibitory effect of adamantylGb3, we investigated whether adamantylGb3 is able to inhibit cell membrane fusion and prevent syncytium formation. NIH 3T3 cells expressing CD4 and either CXCR4 or CCR5 HIV co-receptors, and HeLa cells expressing either HIV-1 X4 or R5 gp120–gp41 or HIV-2 envelope proteins, were co-cultured with or without adamantylGb3. Dose-dependent adamantylGb3 inhibition (> 95%) of fusion/syncytium formation, dependant on gp120 source and chemokine receptor class, was observed for the HIV-1IIIB, the HIV-1Ba-L and the HIV-2 model systems (Fig. 4a–h).
T cells express Gb3 following activation
Although Gb3 is a B cell differentiation antigen , we questioned whether adamantylGb3 might compete for a Gb3 function in T cell HIV infection. Therefore, we monitored the effect of PBMC T-cell activation on GSL synthesis. Following PHA/IL-2 activation, when approximately 95% of cells are T-cell blasts , a major increase in neutral GSL synthesis was observed (Fig. 5). The ceramide trihexoside species was completely susceptible to αgalactosidase (Fig. 5a) indicating that PHA/IL-2 activates T cell globo-series GSL synthesis. Gb3 was detected in the activated T cell GSL extract by VT1 binding (Fig. 5b).
Our results indicate that functional soluble Gb3 analogues, such as the adamantylGb3 we have made, are an effective, novel means to prevent HIV infection in vitro, irrespective of the HIV strain, tropism or CD4-expressing target cell. This mechanism probably involves the inhibition of binding and viral–host cell fusion via the post-CD4 binding, V3 loop/GSL-dependent event, described by Nehete et al. . Lipid raft GSL binding by gp120 facilitates the ability to bind the sparsely distributed CD4 and chemokine coreceptor simultaneously, because overexpression of these receptor components at high density on the cell surface removes the requirement for GSL binding . Nevertheless, the selective inhibition of cell fusion by adamantylGb3 implies additional undefined functions, which may concern the gp120/gp41 conformational change necessary for fusion . Inhibition of HIV-induced cell fusion is the most recent successful therapeutic approach . The fact that pre-incubation of virus with adamantylGb3 is effective in inhibiting HIV infection suggests that adamantylGb3 also acts as a competitor to prevent interactions with target cell GSL prior to fusion. Thus, adamantylGb3 inhibition may be twofold, both preventing fusion and competing for gp120 interactions. Preincubation of target cells with adamantylGb3 was ineffective, indicating virus/adamantylGb3 binding is of central importance. The transient decrease in cell surface CD4 expression seen for cells treated with adamantlyGb3 alone for 1 h is unlikely to contribute to the inhibition, since infection occurs rapidly, within 5–10 min [33,34]. We confirmed this by electron microscopy, where intracellular virus was observed within 20 min of viral addition. Furthermore, as CD4 levels recover, this effect should not play any role, for example in secondary infections which propagate p24gag antigen levels.
Following HIV infection, the HIV protein Nef is responsible for initial down-regulation of CD4  facilitating viral egress, and Nef has also been found to selectively interfere with intracellular Gb3 trafficking , implying that Gb3 might also play a role in Nef function. Intracellular Gb3 trafficking is dependent on lipid rafts  and Nef interactions share this dependence . CD4, CXCR4 and CCR5  are all found within such GSL-enriched lipid rafts, and provide further potential targets for interference by adamantylGb3, which can partition into membranes.
The potential multiple action sites for adamantylGb3 would imply the infrequent development of viral resistance to adamantylGb3. An HIV strain resistant to multiple inhibitors of reverse transcriptase and a protease inhibitor resistant strain demonstrate susceptibility to adamantylGb3, suggesting that therapy based on this approach could be complementary to current approaches. The inhibitory efficacy of adamantylGb3 was lower than our gp120 binding studies  predicted and the effective range (100–300 μM), difficult to maintain clinically. The efficacy is in part, limited by the ability of adamantylGb3 to partition into membranes. Further modification to decrease hydrophobicity should increase specific activity. Nevertheless our studies identify a new target for inhibition of the HIV infection cycle. AdamantylGb3 within a cream, for example, might provide a topical ointment for the prevention of mucosal HIV infection. Such an approach could have far reaching implications for the developing world, where HAART is not readily available.
We show that activated T cells transiently express Gb3, consistent with the aberrant synthesis of Gb3 in T cells from HIV patients . Thus, Gb3 may play a (adamantylGb3 inhibited) role in HIV T-cell infection. This role may not be exclusive, since lymphocytes of p blood group individuals (who lack the αgalactosyltransferase necessary to make Gb3) are susceptible to X4 HIV infection [11,13]. In these T cells, there is a compensatory increase in GM3 ganglioside which may substitute for Gb3 function [11,13], suggesting that GM3 analogues might also prove effective inhibitors of HIV infection. Furthermore, we have shown that PBMC from Fabry patients, in which Gb3 accumulates due to a defect in breakdown, are resistant to R5 but not X4 HIV infection in vitro . In Fabry PHA activated PBMCs resistant to R5 infection, the level of CCR5 is reduced, implicating a link between endogenous Gb3 and chemokine receptor trafficking. Chemokine receptor levels are, however if anything, increased in adamantylGb3 treated cells indicating a different mechanism of inhibiting infection.
HIV infection is mediated by the viral fusion glycoprotein gp120–gp41 binding CD4 , which is the basis of the viral targeting of T lymphocytes and monocyte-macrophages. In addition, it has long been appreciated that the virus can infect CD4-negative cells, primarily those of the brain and gastrointestinal tract. This has spurred the search for alternate HIV receptors that now includes DC-SIGN of dendritic cells , (which interestingly, also express Gb3 ) as well as VPAC1, a receptor for vasoactive intestinal peptide which shows some sequence similarity to gp120 . The partition of these, and the traditional HIV receptors, CD4 and chemokine co-receptors, into GSL enriched lipid rafts [39,43] provides a rationale for gp120/GSL binding, to bridge receptors/co-receptors which may be in separate rafts . Such binding also provides a possible basis for the infection of CD4 negative cells [44,45]. Interference of the gp120 interactions with these various host cell membrane factors is a potentially powerful strategy for the treatment and/or prevention of HIV/AIDS, and may be particularly important in the development of microbicidals.
Infection with HIV and AIDS continues to increase worldwide, despite intense research to control its spread . Current therapies to treat infection that target the virus, such as HAART and vaccine development, have not yet achieved expectations due to drug resistance and viral genetic variance, respectively [47–49]. Alternative therapies are desperately needed. We have shown that a soluble analogue of the glycolipid Gb3, adamantylGb3, binds with high affinity to gp120 and can inhibit in vitro fusion and infection of cell lines and primary cell targets, regardless of the tropism of the virus and resistance to other treatments. Further experiments in in vivo models will be required to determine whether adamantylGb3 can provide a new basis for the prevention and treatment of HIV-1 and HIV-2 infections.
This work was funded by the Canadian Blood Services via a graduate studentship (to N. L.) a grant from the ‘Skate the Dream’ Fund of the Toronto General and Western Hospital Foundation (to D.R.B.), an HSC Foundation studentship (to D.C.), an Ontario HIV Treatment Network small operating grant for Emerging Technologies (to C.A.L. and D.R.B.) and CIHR grant #MT13073 (to C.A.L.) and by the Intramural Research Program of the NIH, NCI. (AP and RB).
Note: Donald R. Branch and Clifford A. Lingwood made equal contributions to this work.
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