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. 2011 Nov 1:4:474.
doi: 10.1186/1756-0500-4-474.

Antiviral mode of action of bovine dialyzable leukocyte extract against human immunodeficiency virus type 1 infection

Humberto H Lara  1 , Liliana Ixtepan-Turrent, Elsa N Garza-Treviño, Jose I Badillo-Almaraz, Cristina Rodriguez-Padilla
Affiliations

Antiviral mode of action of bovine dialyzable leukocyte extract against human immunodeficiency virus type 1 infection

Humberto H Lara et al. BMC Res Notes. .

Abstract

Background: Bovine dialyzable leukocyte extract (bDLE) is derived from immune leukocytes obtained from bovine spleen. DLE has demonstrated to reduce transcription of Human Immunodeficiency Virus Type 1 (HIV-1) and inactivate the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. Therefore, we decided to clarify the mode of antiviral action of bDLE on the inhibition of HIV-1 infection through a panel of antiviral assays.

Results: The cytotoxicity, HIV-1 inhibition activity, residual infectivity of bDLE in HIV-1, time of addition experiments, fusion inhibition of bDLE for fusogenic cells and the duration of cell protection even after the removal of bDLE were all assessed in order to discover more about the mode of the antiviral action.HIV-1 infectivity was inhibited by bDLE at doses that were not cytotoxic for HeLa-CD4-LTR-β-gal cells. Pretreatment of HIV-1 with bDLE did not decrease the infectivity of these viral particles. Cell-based fusion assays helped to determine if bDLE could inhibit fusion of Env cells against CD4 cells by membrane fusion and this cell-based fusion was inhibited only when CD4 cells were treated with bDLE. Infection was inhibited in 80% compared with the positive (without EDL) at all viral life cycle stages in the time of addition experiments when bDLE was added at different time points. Finally, a cell-protection assay against HIV-1 infection by bDLE was performed after treating host cells with bDLE for 30 minutes and then removing them from treatment. From 0 to 7 hours after the bDLE was completely removed from the extracellular compartment, HIV-1 was then added to the host cells. The bDLE was found to protect the cells from HIV-1 infection, an effect that was retained for several hours.

Conclusions: bDLE acted as an antiviral compound and prevented host cell infection by HIV-1 at all viral life cycle stages. These cell protection effects lingered for hours after the bDLE was removed. Interestingly, bDLE inhibited fusion of fusogenic cells by acting only on CD4 cells. bDLE had no virucidal effect, but could retain its antiviral effect on target cells after it was removed from the extracellular compartment, protecting the cells from infection for hours.bDLE, which has no reported side effects or toxicity in clinical trials, should therefore be further studied to determine its potential use as a therapeutic agent in HIV-1 infection therapy, in combination with known antiretrovirals.

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Figures

Figure 1
Figure 1
Cytotoxicity assessment of bDLE and HIV-1 inhibition activity. A) HeLa-CD4-LTR-β-gal cells (5 × 104 cells/well) and B) HIV-1IIIB cell-free viruses (MOI 0.2-0.5) were challenged with two-fold serial dilutions of bDLE. Cell viability and β-gal activity were measured with a luciferase-based assay 24 h after nanosilver exposure. Percentage values are relative to the positive control (no compound treatment). The data represent the means ± standard deviations from three separate experiments, each of which was carried out in duplicate.
Figure 2
Figure 2
Residual activity in HIV-1 strains. HIV-1IIIB cell-free viruses were exposed to serial dilutions of bDLE for 5 minutes. The viruses were then ultracentrifuged, washed twice and added to HeLa-CD4-LTR-β-gal cells. After 24 hours, β-gal activity was measured. Percentage values are relative to the positive control (infected cells without bDLE treatment). The data represent the means ± standard deviations from three separate experiments, each of which was carried out in duplicate.
Figure 3
Figure 3
Inhibition of Env/CD4-mediated membrane fusion. β-gal activity was measured after CD4 cells and Env cells were co-cultured after exposure to A) bDLE, B) UC781 and C) T-20 under different circumstances: (■) CD4 cells were exposed to the compound and co-cultured with Env cells for 24 hours; (▲) CD4 cells were exposed to the compound for 30 minutes, washed, and co-cultured with Env cells for 24 hours; (✘) Env cells were exposed to the compound for 30 minutes, washed, and co-cultured with CD4 cells for 24 hours. Percentage values are relative to the positive control (cell-to-cell fusion without pretreatment with drug). The data represent the means ± standard deviations from three separate experiments, each of which was carried out in duplicate.
Figure 4
Figure 4
Time of intervention in HIV-1 life cycle. HeLa/CD4-LTR-β-gal cells were infected with HIV-1IIIB cell-free virus before A) bDLE (2 IU), B) T-20 (100 μM), C) UC781 (70 nM), D) 118-D-24 (120 μM) and E) Amprenavir (0.1 mM), were added upon HIV-1 inoculation (time zero) or at various time points post-infection. β-gal activity was measured following 24 hr of incubation. Percentage values are relative to the positive control (infected cells without drug pretreatment). The data represent the means ± standard deviations from three separate experiments, each of which was carried out in duplicate.
Figure 5
Figure 5
Cell protection against HIV-1 infection. β-gal activity was measured after HeLa/CD4-LTR-β-gal cells were exposed to bDLE (2 IU) for 30 minutes, washed and exposed to HIV-1IIIB cell-free virus at various time points post-treatment. Percentage values are relative to the positive control (infected cells without drug pretreatment). The data represent the means ± standard deviations from three separate experiments, each of which was carried out in duplicate.
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