Inhibition of highly productive HIV-1 infection in T cells, primary human macrophages, microglia, and astrocytes by Sargassum fusiforme

Abstract

Background: The high rate of HIV-1 mutation and increasing resistance to currently available antiretroviral (ART) therapies highlight the need for new antiviral agents. Products derived from natural sources have been shown to inhibit HIV-1 replication during various stages of the virus life cycle, and therefore represent a potential source of novel therapeutic agents. To expand our arsenal of therapeutics against HIV-1 infection, we investigated aqueous extract from Sargassum fusiforme (S. fusiforme) for ability to inhibit HIV-1 infection in the periphery, in T cells and human macrophages, and for ability to inhibit in the central nervous system (CNS), in microglia and astrocytes.

Results: S. fusiforme extract blocked HIV-1 infection and replication by over 90% in T cells, human macrophages and microglia, and it also inhibited pseudotyped HIV-1 (VSV/NL4-3) infection in human astrocytes by over 70%. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5)-tropic HIV-1, was dose dependant and long lasting, did not inhibit cell growth or viability, was not toxic to cells, and was comparable to inhibition by the nucleoside analogue 2', 3'-didoxycytidine (ddC). S. fusiforme treatment blocked direct cell-to-cell infection spread. To investigate at which point of the virus life cycle this inhibition occurs, we infected T cells and CD4-negative primary human astrocytes with HIV-1 pseudotyped with envelope glycoprotein of vesicular stomatitis virus (VSV), which bypasses the HIV receptor requirements. Infection by pseudotyped HIV-1 (VSV/NL4-3) was also inhibited in a dose dependant manner, although up to 57% less, as compared to inhibition of native NL4-3, indicating post-entry interferences.

Conclusion: This is the first report demonstrating S. fusiforme to be a potent inhibitor of highly productive HIV-1 infection and replication in T cells, in primary human macrophages, microglia, and astrocytes. Results with VSV/NL4-3 infection, suggest inhibition of both entry and post-entry events of the virus life cycle. Absence of cytotoxicity and high viability of treated cells also suggest that S. fusiforme is a potential source of novel naturally occurring antiretroviral compounds that inhibit HIV-1 infection and replication at more than one site of the virus life cycle.

Figures

Figure 1

Analysis of growth kinetics and viability in T cells treated with S. fusiforme. 1G5 T cells were treated with 2 mg/ml or 4 mg/ml S. fusiforme, or with 10-6 M ddC, or were mock treated. (A) Total cell number, and (B) % viable cells from total, was monitored at the indicated time points after infection, by trypan blue exclusion assay by counting at least 200 cells each in three different fields under ×20 magnification using an Olympus BH-2 fluorescence microscope. Experiment was repeated with primary human PBMC's treated with 1.5, 3, or 4.5 mg/ml S. fusiforme, or with 10-6 M ddC, or mock treated, and measured (C) Total cell number, and (D) % viable cells from total. PBMC's experiments are representative of 3 separate experiments, with SEM less than 5% (not shown).

Figure 2

Dose response of HIV-1 inhibition and cell viability in T cells treated with S. fusiforme. 1G5 T cells were treated for 24 h with increasing concentrations of S. fusiforme, or with 10-6 M ddC, as indicated; then infected with CXCR4 tropic HIV-1 (NL4-3) at multiplicity of infection (moi) of 0.01 for 1.5 h, washed 3 times, and returned to culture with same concentrations of each treatment for the duration of the experiment. (A) On day 3 after infection, intracellular luciferase gene marker expression was measured from cell lysates adjusted to same number of viable cells by MTT. Percent inhibition of HIV-1 was calculated utilizing formula in the Methods section, and plotted on the Y-axis as % Inhibition. In parallel, (B) cell viability for each treatment was quantified by MTT uptake, measured at 570 nm absorbance. Data are mean +/- SD of triplicates. Representative of three separate experiments.

Figure 3

Time course of HIV-1 inhibition and viability in T cells. 1G5 T cells were 24 h treated with either 2 mg/ml S. fusiforme, or with 10-6 M ddC; then infected with NL4-3 at 0.01 moi for 1.5 h, washed 3 times, and returned to culture with same concentration of each treatment for the duration of the experiment. On day 3 post-infection, (A) gene expression of intracellular luciferase was measured from cell lysates adjusted to same number of viable cells, and % inhibition calculated and plotted on the Y-axis. Data are mean +/- SD of triplicates. In parallel, (B) cell viability was determined by trypan blue exclusion assay by counting at least 200 cells each, in three different fields under ×20 magnification using an Olympus BH-2 fluorescence microscope.

Figure 4

Inhibition of cell-to-cell infection and syncytia formation. Uninfected 1G5 T cells were pretreated for 24 h with either (A) mock, (B) 10-6 M ddC, or with ddC and (B) 2 mg/ml or (C) 4 mg/ml S. fusiforme, or with S. fusiforme only at (D) 2 mg/ml or (E) 4 mg/ml. 1G5 cells were cocultivated at 1:1 ratio with CEM cells that were infected with NL4-3 at 0.01 moi. 24 h after cocultivation, cells were examined for syncytium formation using Leica DM IL Fluo microscope, ×20 magnification (A-F). Cell cultures were monitored for luciferase expression, and % inhibition was calculated from maximal luciferase expression from untreated 1G5 cells (1.9 × 105 RLU, not shown), which was plotted and is indicated on top of each bar (H). Data are mean +/- SD of triplicates. Uninfected adherent GHOST [29] cells were ddC treated and cocultivated at 1:1 ratio with HIV infected 1G5 cells for 24 h, and examined for syncytia formation by green fluorescence (G). Image shows fluorescence micrograph taken of a green fluorescent giant cell, which was superimposed on the same field phase contrast black and white image.

Figure 5

Inhibition of HIV-1 expression in human macrophages and microglia. Either, (A) human macrophages or (B) human fetal microglia were 24 h treated with 1 mg/ml S. fusiforme, or with 10-6 M ddC, infected with primary CCR5-tropic isolate ADA at 0.2 pg of p24/cell for 2 h, washed 3 times, and returned to culture with same concentration of each treatment for the duration of the experiment. At the indicated time points after infection HIV-1 expression was monitored by p24 production in cell-free supernatants by ELISA, % inhibition calculated as described in Methods and plotted on the y-axis. Data are mean +/- SD of triplicates. Representative of 2 experiments.

Figure 6

Inhibition of infection with pseudotyped HIV-1 in T cells andhuman astrocytes. (A) 1G5 T cells were treated with increasing concentrations of S. fusiforme and infected with either NL4-3 at 0.01 moi or with VSV/NL4-3 at 0.005 moi. 3 days after infection, % inhibition was calculated from luciferase expression from cell lysates adjusted to same number of viable cells by MTT. (B) Human fetal CD4 negative astrocytes were treated with 1 mg/ml S. fusiforme, or with 10-6 M ddC, infected with VSV/NL4-3 at 0.4 moi, and infection kinetics monitored by p24 expression in cell free supernatants at the indicated time points post infection. Data are mean +/- SD of triplicates. Representative of 2 experiments.