Author Correction: Cardiac glycoside/aglycones inhibit HIV-1 gene expression by a mechanism requiring MEK1/2-ERK1/2 signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cardiac glycoside/aglycones inhibit HIV-1 gene expression by a mechanism requiring MEK1/2-ERK1/2 signaling.

The capacity of HIV-1 to develop resistance to current drugs calls for innovative strategies to control this infection. We aimed at developing novel inhibitors of HIV-1 replication by targeting viral RNA processing-a stage dependent on conserved host processes. We previously reported that digoxin is a potent inhibitor of this stage. Herein, we identify 12 other cardiac glycoside/aglycones or cardiotonic steroids (CSs) that impede HIV growth in HIV-infected T cells from clinical patients at IC50s (1.1-1.3 nM) that are 2-26 times below concentrations used in patients with heart conditions. We subsequently demonstrate that CSs inhibit HIV-1 gene expression in part through modulation of MEK1/2-ERK1/2 signaling via interaction with the Na+/K+-ATPase, independent of alterations in intracellular Ca2+. Supporting this hypothesis, depletion of the Na+/K+-ATPase or addition of a MEK1/2-ERK1/2 activator also impairs HIV-1 gene expression. Similar to digoxin, all CSs tested induce oversplicing of HIV-1 RNAs, reducing unspliced (Gag) and singly spliced RNAs (Env/p14-Tat) encoding essential HIV-1 structural/regulatory proteins. Furthermore, all CSs cause nuclear retention of genomic/unspliced RNAs, supporting viral RNA processing as the underlying mechanism for their disruption of HIV-1 replication. These findings call for further in vivo validation and supports the targeting of cellular processes to control HIV-1 infection.

Glycosphingolipid storage in Fabry mice extends beyond globotriaosylceramide and is affected by ABCB1 depletion.

AIM: Fabry disease is caused by α-galactosidase A deficiency leading to accumulation of globotriaosylceramide (Gb3) in tissues. Clinical manifestations do not appear to correlate with total Gb3 levels. Studies examining tissue distribution of specific acyl chain species of Gb3 and upstream glycosphingolipids are lacking. MATERIAL & METHODS/RESULTS: Thorough characterization of the Fabry mouse sphingolipid profile by LC-MS revealed unique Gb3 acyl chain storage profiles. Storage extended beyond Gb3; all Fabry tissues also accumulated monohexosylceramides. Depletion of ABCB1 had a complex effect on glycosphingolipid storage. CONCLUSION: These data provide insights into how specific sphingolipid species correlate with one another and how these correlations change in the α-galactosidase A-deficient state, potentially leading to the identification of more specific biomarkers of Fabry disease.

Inhibition of Rab prenylation by statins induces cellular glycosphingolipid remodeling.

Statins, which specifically inhibit HMG Co-A reductase, the rate-limiting step of cholesterol biosynthesis, are widely prescribed to reduce serum cholesterol and cardiac risk, but many other effects are seen. We now show an effect of these drugs to induce profound changes in the step-wise synthesis of glycosphingolipids (GSLs) in the Golgi. Glucosylceramide (GlcCer) was increased several-fold in all cell lines tested, demonstrating a widespread effect. Additionally, de novo or elevated lactotriaosylceramide (Lc3Cer; GlcNAcß1-3Galß1-4GlcCer) synthesis was observed in 70%. Western blot showed that GlcCer synthase (GCS) was elevated by statins, and GCS and Lc3Cer synthase (Lc3S) activities were increased; however, transcript was elevated for Lc3S only. Supplementation with the isoprenoid precursor, geranylgeranyl pyrophosphate (GGPP), a downstream product of HMG Co-A reductase, reversed statin-induced glycosyltransferase and GSL elevation. The Rab geranylgeranyl transferase inhibitor 3-PEHPC, but not specific inhibitors of farnesyl transferase, or geranylgeranyl transferase I, was sufficient to replicate statin-induced GlcCer and Lc3Cer synthesis, supporting a Rab prenylation-dependent mechanism. While total cholesterol was unaffected, the trans-Golgi network (TGN) cholesterol pool was dissipated and medial Golgi GCS partially relocated by statins. GSL-dependent vesicular retrograde transport of Verotoxin and cholera toxin to the Golgi/endoplasmic reticulum were blocked after statin or 3-PEHPC treatment, suggesting aberrant, prenylation-dependent vesicular traffic as a basis of glycosyltransferase increase and GSL remodeling. These in vitro studies indicate a previously unreported link between Rab prenylation and regulation of GCS activity and GlcCer metabolism.

Cholesterol masks membrane glycosphingolipid tumor-associated antigens to reduce their immunodetection in human cancer biopsies.

Glycosphingolipids (GSLs) are neoplastic and normal/cancer stem cell markers and GSL/cholesterol-containing membrane rafts are increased in cancer cell plasma membranes. We define a novel means by which cancer cells can restrict tumor-associated GSL immunoreactivity. The GSL-cholesterol complex reorients GSL carbohydrate to a membrane parallel, rather than perpendicular conformation, largely unavailable for antibody recognition. Methyl-ß-cyclodextrin cholesterol extraction of all primary human tumor frozen sections tested (ovarian, testicular, neuroblastoma, prostate, breast, colon, pheochromocytoma and ganglioneuroma), unmasked previously "invisible" membrane GSLs for immunodetection. In ovarian carcinoma, globotriaosyl ceramide (Gb3), the GSL receptor for the antineoplastic Escherichia coli-derived verotoxin, was increased throughout the tumor. In colon carcinoma, Gb3 detection was vastly increased within the neovasculature and perivascular stroma. In tumors considered Gb3 negative (neuroblastoma, Leydig testicular tumor and pheochromocytoma), neovascular Gb3 was unmasked. Tumor-associated GSL stage-specific embryonic antigen (SSEA)-1, SSEA-3, SSEA-4 and globoH were unmasked according to tumor: SSEA-1 in prostate/colon; SSEA-3 in prostate; SSEA-4 in pheochromocytoma/some colon tumors; globoH in prostate/some colon tumors. In colon, anti-SSEA-1 was tumor cell specific. Within the GSL-cholesterol complex, filipin-cholesterol binding was also reduced. These results may relate to the ill-defined benefit of statins on cancer prognosis, for example, prostate carcinoma. We found novel anti-tumor GSL antibodies circulating in 3/5 statin-treated, but not untreated, prostate cancer patients. Lowering tumor membrane cholesterol may permit immune recognition of otherwise unavailable tumor-associated GSL carbohydrate, for more effective immunosurveillance and active/passive immunotherapy. Our results show standard immunodetection of tumor GSLs significantly under assesses tumor membrane GSL content, impinging on the current use of such antigens as cancer vaccines.

CD4(+) T-cells are unable to express the HIV natural resistance factor globotriosylceramide.

Globotriaosylceramide (Gb(3)) is a cell surface-expressed natural resistance factor for HIV infection, but, its expression in human T-cells remains unknown. Therefore, Gb(3) in resting or activated CD4(+) T-cells was assessed by flow cytometry and thin layer chromatography of cell extracts. We found the majority of CD4(+) T-cells, whether resting or activated, do not express Gb(3) at significant levels (<2% positive cells). Thus, HIV treatment or prevention strategies must focus on development of soluble Gb(3) analogues for inhibition of HIV infection.

Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane.

The influenza virus (IFV) acquires its envelope by budding from host cell plasma membranes. Using quantitative shotgun mass spectrometry, we determined the lipidomes of the host Madin-Darby canine kidney cell, its apical membrane, and the IFV budding from it. We found the apical membrane to be enriched in sphingolipids (SPs) and cholesterol, whereas glycerophospholipids were reduced, and storage lipids were depleted compared with the whole-cell membranes. The virus membrane exhibited a further enrichment of SPs and cholesterol compared with the donor membrane at the expense of phosphatidylcholines. Our data are consistent with and extend existing models of membrane raft-based biogenesis of the apical membrane and IFV envelope.

Comparison of detection methods for cell surface globotriaosylceramide.

The cell surface-expressed glycosphingolipid (GSL), globotriaosylceramide (Gb(3)), is becoming increasingly important and is widely studied in the areas of verotoxin (VT)-mediated cytotoxicity, human immunodeficiency virus (HIV) infection, immunology and cancer. However, despite its diverse roles and implications, an optimized detection method for cell surface Gb(3) has not been determined. GSLs are differentially organized in the plasma membrane which can affect their availability for protein binding. To examine various detection methods for cell surface Gb(3), we compared four reagents for use in flow cytometry analysis. A natural ligand (VT1B) and three different monoclonal antibodies (mAbs) were optimized and tested on various human cell lines for Gb(3) detection. A differential detection pattern of cell surface Gb(3) expression, which was influenced by the choice of reagent, was observed. Two mAb were found to be suboptimal. However, two other methods were found to be useful as defined by their high percentage of positivity and mean fluorescence intensity (MFI) values. Rat IgM anti-Gb(3) mAb (clone 38-13) using phycoerythrin-conjugated secondary antibody was found to be the most specific detection method while the use of VT1B conjugated to Alexa488 fluorochrome was found to be the most sensitive; showing a rare crossreactivity only when Gb(4) expression was highly elevated. The findings of this study demonstrate the variability in detection of Gb(3) depending on the reagent and cell target used and emphasize the importance of selecting an optimal methodology in studies for the detection of cell surface expression of Gb(3).

Glycosphingolipid functions.

The combination of carbohydrate and lipid generates unusual molecules in which the two distinctive halves of the glycoconjugate influence the function of each other. Membrane glycolipids can act as primary receptors for carbohydrate binding proteins to mediate transmembrane signaling despite restriction to the outer bilayer leaflet. The extensive heterogeneity of the lipid moiety plays a significant, but still largely unknown, role in glycosphingolipid function. Potential interplay between glycolipids and their fatty acid isoforms, together with their preferential interaction with cholesterol, generates a complex mechanism for the regulation of their function in cellular physiology.

Cholesterol modulates glycolipid conformation and receptor activity.

We document a new dimension of surface recognition in which communication is controlled through the collective behavior of lipids. Membrane cholesterol induces a tilt in glycolipid receptor headgroup, resulting in loss of access for ligand binding. This property appears to organize erythrocyte blood group presentation and glycolipid receptor function during the activation of sperm fertility, suggesting that lipid 'allostery' is a means to regulate membrane recognition processes.

The role of glycosphingolipids in HIV/AIDS.

Much remains unknown about basic aspects of HIV-1 infection and cell susceptibility. Glycosphingolipid (GSL) binding by the HIV-1 adhesin gp120 has long been implicated in the infection of non-lymphoid cells, as well as CD4(+) T cells and monocytes, the primary targets of HIV-1 infection. We have identified the P(k) blood group antigen (a GSL) globotriaosylceramide (Gb(3)) as a new resistance effector against HIV-1 infection. Significantly, the α-galactosyltransferase (A4GALT, Gb(3) synthase) responsible for the synthesis of Gb(3) is included among markers genetically linked to HIV-1 resistance. Other GSLs, including GalCer and GM3, have been implicated as facilitators of HIV infection. This review will address the role of GSLs in HIV/AIDS but focus on the role of Gb(3) as a newly described natural resistance factor for the prevention of HIV infection and examine potential therapies that would utilize soluble analogues of this unique GSL.

A major fraction of glycosphingolipids in model and cellular cholesterol-containing membranes is undetectable by their binding proteins.

Glycosphingolipids (GSLs) accumulate in cholesterol-enriched cell membrane domains and provide receptors for protein ligands. Lipid-based "aglycone" interactions can influence GSL carbohydrate epitope presentation. To evaluate this relationship, Verotoxin binding its receptor GSL, globotriaosyl ceramide (Gb(3)), was analyzed in simple GSL/cholesterol, detergent-resistant membrane vesicles by equilibrium density gradient centrifugation. Vesicles separated into two Gb(3/)cholesterol-containing populations. The lighter, minor fraction (<5% total GSL), bound VT1, VT2, IgG/IgM mAb anti-Gb(3), HIVgp120 or Bandeiraea simplicifolia lectin. Only IgM anti-Gb(3), more tolerant of carbohydrate modification, bound both vesicle fractions. Post-embedding cryo-immuno-EM confirmed these results. This appears to be a general GSL-cholesterol property, because similar receptor-inactive vesicles were separated for other GSL-protein ligand systems; cholera toxin (CTx)-GM1, HIVgp120-galactosyl ceramide/sulfatide. Inclusion of galactosyl or glucosyl ceramide (GalCer and GlcCer) rendered VT1-unreactive Gb(3)/cholesterol vesicles, VT1-reactive. We found GalCer and GlcCer bind Gb(3), suggesting GSL-GSL interaction can counter cholesterol masking of Gb(3). The similar separation of Vero cell membrane-derived vesicles into minor "binding," and major "non-binding" fractions when probed with VT1, CTx, or anti-SSEA4 (a human GSL stem cell marker), demonstrates potential physiological relevance. Cell membrane GSL masking was cholesterol- and actin-dependent. Cholesterol depletion of Vero and HeLa cells enabled differential VT1B subunit labeling of "available" and "cholesterol-masked" plasma membrane Gb(3) pools by fluorescence microscopy. Thus, the model GSL/cholesterol vesicle studies predicted two distinct membrane GSL formats, which were demonstrated within the plasma membrane of cultured cells. Cholesterol masking of most cell membrane GSLs may impinge many GSL receptor functions.

A synthetic globotriaosylceramide analogue inhibits HIV-1 infection in vitro by two mechanisms.

Previously, it was shown that the cell-membrane-expressed glycosphingolipid, globotriaosylceramide (Gb(3)/P(k)/CD77), protects against HIV-1 infection and may be a newly described natural resistance factor against HIV infection. We have now investigated the potential of a novel, water soluble, non-toxic and completely synthetic analogue of Gb(3)/P(k) (FSL-Gb(3)) to inhibit HIV-1 infection in vitro. A uniquely designed analogue, FSL-Gb(3), of the natural Gb(3)/P(k) molecule was synthesized. HIV-1(IIIB) (X4 virus) and HIV-1(Ba-L) (R5 virus) infection of PHA/interleukin-2-activated, peripheral blood mononuclear cells (PBMCs) and Jurkat T cells in vitro was assessed, as well as infection of U87.CD4.CCR5 by various clinical R5 tropic viruses after treatment with FSL-Gb(3). We monitored Gb(3), CD4 and CXCR4 expression by fluorescent antibody cell sorting and viral replication by p24(gag) ELISA. Total cellular Gb(3) was examined by glycosphingolipid extraction and thin layer chromatography. In vivo toxicity was monitored in mice by histological assessment of vital organs and lymphoid tissue. FSL-Gb(3) blocked X4 and R5 of both lab and clinical viral strains in activated PBMCs or the U87.CD4.CCR5 cell line with a 50% inhibitory concentration (IC(50)) of approximately 200-250 microM. FACS and TLC overlay showed that FSL-Gb(3) can insert itself into cellular plasma membranes and that cellular membrane-absorbed FSL-Gb(3) is able to inhibit subsequent HIV-1 infection. There was no effect of FSL-Gb(3) on cell surface levels of CD4 or CXCR4. Thus, FSL-Gb(3) can inhibit HIV-1 by two mechanisms: direct inhibition of virus and inhibition of viral entry. Infusion of FSL-Gb(3) into laboratory mice at doses well in excess of theoretical therapeutic doses was tolerated with no untoward reactions. Our results demonstrate the potential utility of using a completely synthetic, water soluble globotriaosylceramide analogue, FSL-Gb(3), having low toxicity, for possible future use as a novel therapeutic approach for the systemic treatment of HIV/AIDS.

New aspects of the regulation of glycosphingolipid receptor function.

We propose that the fatty acid heterogeneity of glycosphingolipids may compensate for the relative few and simple glycosphingolipid structures found in mammalian cells. Variation in GSL fatty acid composition may mediate aglycone regulation of GSL membrane receptor function by a differential interaction with cholesterol and other membrane components which may be differentially organized within plasma membrane lipid domains. These concepts are specifically illustrated in model membrane studies and in relation to the role of the glycolipid, globotriaosyl ceramide (Gb(3)) in verotoxin-induced renal pathology and gp120 binding in HIV infection.

A soluble sulfogalactosyl ceramide mimic promotes Delta F508 CFTR escape from endoplasmic reticulum associated degradation.

AdaSGC binds Hsc70s to inhibit ATPase activity. Using single-turnover assays, adaSGC, a soluble SGC mimic, preferentially inhibited Hsp40-activated Hsc70 ATP hydrolysis (Ki approximately 10 microM) to reduce C-terminal Hsc70-peptide binding and, potentially, chaperone function. ERAD of misfolded Delta F508 CFTR requires Hsc70-Hsp40 chaperones. In transfected baby hamster kidney (BHK) cells, adaSGC increased Delta F508CFTR ERAD escape, and after low-temperature glycerol rescue, maturation, and iodide efflux. Inhibition of SGC biosynthesis reduced Delta F508CFTR but not wtCFTR expression, whereas depletion of other glycosphingolipids had no affect. WtCFTR transfected BHK cells showed increased SGC synthesis compared with Delta F508CFTR/mock-transfected cells. Partial rescue of Delta F508CFTR by low-temperature glycerol increased SGC synthesis. AdaSGC also increased cellular endogenous SGC levels. SGC in the lung, liver, and kidney was severely depleted in Delta F508CFTR compared with wtCFTR mice, suggesting a role for CFTR in SGC biosynthesis.

Detergent-resistant globotriaosyl ceramide may define verotoxin/glomeruli-restricted hemolytic uremic syndrome pathology.

Verotoxin binding to its receptor, globotriaosyl ceramide(Gb(3)) mediates the glomerular pathology of hemolytic uremic syndrome, but Gb(3) is expressed in both tubular and glomerular cells. Gb(3) within detergent-resistant membranes, an index of glycolipid-cholesterol enriched lipid rafts, is required for in vitro cytotoxicity. We found that verotoxin 1 and 2 binding to human adult renal glomeruli is detergent resistant, whereas the strong verotoxin binding to renal tubules is detergent sensitive. Verotoxin binding to pediatric glomeruli was detergent resistant but binding to adult glomeruli was enhanced, remarkably for some samples, by detergent extraction. Detergent-sensitive glomerular components may provide age-related protection against verotoxin glomerular binding. Mouse glomeruli remained verotoxin unreactive after detergent extraction, whereas tubular binding was lost. Cholesterol extraction induced strong verotoxin binding in poorly reactive adult glomeruli, suggesting cholesterol can mask Gb(3) in glomerular lipid rafts. Binding of the human immunodeficiency virus (HIV) adhesin, gp120 (another Gb(3) ligand) was detergent sensitive, tubule-restricted, and inhibited by verotoxin B subunit pretreatment, and may relate to HIV nephropathy. Our study shows that differential membrane Gb(3) organization in glomeruli and tubules provides a basis for the age- and glomerular-restricted pathology of hemolytic uremic syndrome.

Inhibition of Simian Virus 40 replication by targeting the molecular chaperone function and ATPase activity of T antigen.

Polyomaviruses such as BK virus and JC virus have been linked to several diseases, but treatments that thwart their propagation are limited in part because of slow growth and cumbersome culturing conditions. In contrast, the replication of one member of this family, Simian Virus 40 (SV40), is robust and has been well-characterized. SV40 replication requires two domains within the viral-encoded large tumor antigen (TAg): The ATPase domain and the N-terminal J domain, which stimulates the ATPase activity of the Hsp70 chaperone. To assess whether inhibitors of polyomavirus replication could be identified, we examined a recently described library of small molecules, some of which inhibit chaperone function. One compound, MAL2-11B, inhibited both TAg's endogenous ATPase activity and the TAg-mediated activation of Hsp70. MAL2-11B also reduced SV40 propagation in plaque assays and compromised DNA replication in cell culture and in vitro. Furthermore, the compound significantly reduced the growth of BK virus in a human kidney cell line. These data indicate that pharmacological inhibition of TAg's chaperone and ATPase activities may provide a route to combat polyomavirus-mediated disease.

Oxidation of the primary hydroxyl group of galactose of galactaosyl ceramide analogue by chemical method-precursors for the synthesis of labeled conjugates.

Isotopic labeling of the C-6 of a model glycosphingolipid (2S, 3R, 4E)-2-(1-adamantanacetamido)-3-hydroxy-4-octadecenyl-beta-D-galactopyranoside, GalCAda, is described. Oxidation of (2S, 3R, 4E)-2-(1-adamantanacetamido)-3-(benzoyloxy)-4-octadecenyl-2,3,4-tri-O-benzoyl-beta-D-galactopyranoside with o-iodoxybenzoic acid gave the dialdoside derivative in good yield. Reduction of the dialdoside with sodium borodeuteride gave the deuterium labeled D-GalCAda, with a cumulative yield of 35%.

The human P(k) histo-blood group antigen provides protection against HIV-1 infection.

Several human histo-blood groups are glycosphingolipids, including P/P1/P(k). Glycosphingolipids are implicated in HIV-host-cell-fusion and some bind to HIV-gp120 in vitro. Based on our previous studies on Fabry disease, where P(k) accumulates and reduces infection, and a soluble P(k) analog that inhibits infection, we investigated cell surface-expressed P(k) in HIV infection. HIV-1 infection of peripheral blood-derived mononuclear cells (PBMCs) from otherwise healthy persons, with blood group P(1)(k), where P(k) is overexpressed, or blood group p, that completely lacks P(k), were compared with draw date-matched controls. Fluorescence-activated cell sorter analysis and/or thin layer chromatography were used to verify P(k) levels. P(1)(k) PBMCs were highly resistant to R5 and X4 HIV-1 infection. In contrast, p PBMCs showed 10- to 1000-fold increased susceptibility to HIV-1 infection. Surface and total cell expression of P(k), but not CD4 or chemokine coreceptor expression, correlated with infection. P(k) liposome-fused cells and CD4(+) HeLa cells manipulated to express high or low P(k) levels confirmed a protective effect of P(k). We conclude that P(k) expression strongly influences susceptibility to HIV-1 infection, which implicates P(k) as a new endogenous cell-surface factor that may provide protection against HIV-1 infection.

Induction of HIV-1 resistance: cell susceptibility to infection is an inverse function of globotriaosyl ceramide levels.

To examine the role of the glycosphingolipid (GSL), globotriaosylceramide (Gb(3), CD77, p(k) blood group antigen) in HIV-1 infection, we have pharmacologically modulated Gb(3) metabolism in an X4 HIV-1 infectable monocytic cell line (THP-1) that naturally expresses Gb(3) and in a Gb(3)-expressing glioblastoma cell line (U87) transfected to express both CD4 and CCR5 to permit R5 HIV-1 infection. THP-1 and U87 cells were treated with either a competitive inhibitor of alpha-galactosidase A, 1-deoxygalactonojirimycin (DGJ) to induce Gb(3) accumulation, or a glucosylceramide synthase inhibitor, phenyl-2-palmitylamino-3-pyrrolidino-1-propanol (P4) to deplete cells of Gb(3). HIV susceptibility was determined via measurement of p24(gag) antigen production by ELISA. In addition, total cellular Gb(3) content was determined using thin layer chromatography followed by Verotoxin1 overlay binding. The cell surface expression of Gb(3) was verified by FACS analysis. We found that DGJ significantly decreased THP-1 and U87 cell susceptibility to HIV-1(IIIB) and HIV-1(BaL) infection, respectively, at a concentration of approximately 100 microM. In contrast, P4 (2 microM) substantially increased cellular susceptibility to HIV-1 infection. Total cellular GSL analysis verified increased Gb(3) expression in cells treated with DGJ and considerable reduction of Gb(3) in P4-treated cells as compared to controls. These results show a reciprocal relationship between Gb(3) expression and infection with either X4 HIV-1(IIIB) or R5 HIV-1(Ba-L). These results support previous studies that Gb(3) provides resistance to HIV infection. Variable Gb(3) expression may provide a natural HIV resistance factor in the general population, and pharmacological manipulation of Gb(3) levels may provide an approach to induction of HIV resistance.