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

Abstract

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.

FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability

“…In addition to the modification of cell and virion surfaces FSL constructs can also be infused directly into the circulation of laboratory animals causing in vivo modification of circulating cells 7 and to inhibit viruses 12 , toxins, 12 and antibodies 7 . FLS constructs have also been used to decorate liposomes and can be printed onto paper surfaces 13 , where they immobilize, and can then be used in diagnostic assays.…”

“…FSL construct functional moieties (F) so far include a range of saccharides including blood group-related determinants, sialic acids, hyaluronan polysaccharides, fluorophores, biotin, radiolabels, and a range of peptides [3][4][5][6][7][8][9][10][11][12] . FSL constructs have been used in modifying embryos, spermatozoa, zebrafish, epithelial/endometrial cells, red blood cells, and virions to create quality controls systems and diagnostic panels, to modify cell adhesion/ interaction/ separation/ immobilization, and for in vitro and in vivo imaging of cells/virions [3][4][5][6][7][8][9][10][11][12] .…”

“…FSL constructs have been used in modifying embryos, spermatozoa, zebrafish, epithelial/endometrial cells, red blood cells, and virions to create quality controls systems and diagnostic panels, to modify cell adhesion/ interaction/ separation/ immobilization, and for in vitro and in vivo imaging of cells/virions [3][4][5][6][7][8][9][10][11][12] .…”

Glycosphingolipids in HIV/AIDS: The Potential Therapeutic Application

“…animal studies have shown no toxicity at millimolar quantities systemically. The unusual tail also allows for this analogue to insert itself into cell membranes and convert an HIV-permissive Gb 3 -negative T-cell into a Gb 3 -positive T-cell that resists HIV infection (Harrison et al, 2010 (Harrison et al, 2010). This was shown both for laboratory strains of HIV-1 as well as for clinical isolates of R5 HIV-1 viruses.…”

“…A completely synthetic water soluble analogue of Gb 3 termed Functional head Spacer Lipid tail-Gb 3 (FSL-Gb 3 ) was shown to inhibit X4 and R5 HIV-1 infection with a similar 50% inhibitory activity (IC 50 ) as adaGb 3 (Harrison et al, 2010). This Gb 3 analogue was unique in that the lipid tails were replaced with phosphatidylethanolamine and a spacer region containing multiple ionic residues allowed for complete solubility in aqueous media.…”