Abstract
Our previous genetic, pharmacological and analogue protection studies identified the glycosphingolipid, Gb3 (globotriaosylceramide, Pk blood group antigen) as a natural resistance factor for HIV infection. Gb3 is a B cell marker (CD77), but a fraction of activated peripheral blood mononuclear cells (PBMCs) can also express Gb3. Activated PBMCs predominantly comprise CD4+ T-cells, the primary HIV infection target. Gb3 is the sole receptor for Escherichia coli verotoxins (VTs, Shiga toxins). VT1 contains a ribosome inactivating A subunit (VT1A) non-covalently associated with five smaller receptor-binding B subunits. The effect of VT on PHA/IL2-activated PBMC HIV susceptibility was determined. Following VT1 (or VT2) PBMC treatment during IL2/PHA activation, the small Gb3+/CD4+ T-cell subset was eliminated but, surprisingly, remaining CD4+ T-cell HIV-1IIIB (and HIV-1Ba-L) susceptibility was significantly reduced. The Gb3-Jurkat T-cell line was similarly protected by brief VT exposure prior to HIV-1IIIB infection. The efficacy of the VT1A subunit alone confirmed receptor independent protection. VT1 showed no binding or obvious Jurkat cell/PBMC effect. Protective VT1 concentrations reduced PBMC (but not Jurkat cell) proliferation by 50%. This may relate to the mechanism of action since HIV replication requires primary T-cell proliferation. Microarray analysis of VT1A-treated PBMCs indicated up regulation of 30 genes. Three of the top four were histone genes, suggesting HIV protection via reduced gene activation. VT blocked HDAC inhibitor enhancement of HIV infection, consistent with a histone-mediated mechanism. We speculate that VT1A may provide a benign approach to reduction of (X4 or R5) HIV cell susceptibility.
Highlights
Verotoxin or Shiga-like toxins are a family of AB5 subunit toxins produced by enterohemorrhagicE. coli (EHEC)
PHA/IL-2-activated peripheral blood mononuclear cells (PBMCs) are over 95% T-cell blasts [17]
This was confirmed using flow cytometry for the PBMC cell cultures used in this study
Summary
Verotoxin or Shiga-like toxins are a family of AB5 subunit toxins produced by enterohemorrhagic. The enzymatic A subunit of VT has been shown to inhibit expression and replication of two bovine retroviruses, bovine leukemia virus (BLV) and bovine immunodeficiency virus (BIV) [9,10,11]. The catalytic activity of the A subunit is required for the inhibition effect since the catalytically inactive mutant VT1A, E167D, was ineffective. The BLV p24 core protein expression was reduced by VT1A treatment. This reduction was only seen in the cell-associated fraction and not in the culture supernatant, which suggests VT1A eliminates BLV infected cells by lysis [12]. Studies with BIV suggest VT inhibits viral production by inducing apoptosis in infected cells [11]. The effect of verotoxin on PBMC HIV infection was investigated
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