HIV-1 integration is inhibited by stimulation of the VPAC2 neuroendocrine receptor

AbstractSuccessful HIV-1 infection requires a number of specific stages leading to integration of the provirus. We previously suggested that members of the VPAC neuroendocrine receptor family may play a role in HIV-1 infection. We now show that stimulation of the VPAC2 receptor with specific agonists provides strong resistance to HIV-1 infection. Daily stimulation of VPAC2, but not VPAC1 or PAC1, resulted in up to 90% inhibition of X4 or R5 productive infections in either cell lines or PBMCs. VPAC2 agonist stimulation had no effect on cell surface co-receptors, the rate of apoptotic cells, or HIV-1 entry or reverse transcription of viral RNA. However, we provide evidence that VPAC2-specific agonists inhibit HIV-1 infection through an inhibitory effect on the ability of the HIV-1 cDNA to integrate into the host DNA. These data reveal that VPAC2 agonists are appropriate candidates for further study as possible treatments aimed at the amelioration of HIV/AIDS

Verotoxin A Subunit Protects Lymphocytes and T Cell Lines against X4 HIV Infection in Vitro

Our previous genetic, pharmacological and analogue protection studies identified the glycosphingolipid, Gb₃ (globotriaosylceramide, P^k 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 <i>Escherichia coli </i>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-1_IIIB (and HIV-1_Ba-L) susceptibility was significantly reduced. The Gb₃^-Jurkat T-cell line was similarly protected by brief VT exposure prior to HIV-1_IIIB 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

Verotoxin A Subunit Protects Lymphocytes and T Cell Lines against X4 HIV Infection in Vitro

toxin

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 (IC50) of approximately 200-250 mu M. 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

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. Pk 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. (Blood. 2009;113:4980-4991