Sargassum fusiforme fraction is a potent and specific inhibitor of HIV-1 fusion and reverse transcriptase

Abstract

Sargassum fusiforme (Harvey) Setchell has been shown to be a highly effective inhibitor of HIV-1 infection. To identify its mechanism of action, we performed bioactivity-guided fractionation on Sargassum fusiforme mixture. Here, we report isolation of a bioactive fraction SP4-2 (S. fusiforme), which at 8 mug/ml inhibited HIV-1 infection by 86.9%, with IC50 value of 3.7 mug. That represents 230-fold enhancement of antiretroviral potency as compared to the whole extract. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5) tropic HIV-1. Specifically, 10 mug/ml SP4-2 blocked HIV-1 fusion and entry by 53%. This effect was reversed by interaction of SP4-2 with sCD4, suggesting that S. fusiforme inhibits HIV-1 infection by blocking CD4 receptor, which also explained observed inhibition of both X4 and R5-tropic HIV-1. SP4-2 also inhibited HIV-1 replication after virus entry, by directly inhibiting HIV-1 reverse transcriptase (RT) in a dose dependent manner by up to 79%. We conclude that the SP4-2 fraction contains at least two distinct and biologically active molecules, one that inhibits HIV-1 fusion by interacting with CD4 receptor, and another that directly inhibits HIV-1 RT. We propose that S. fusiforme is a lead candidate for anti-HIV-1 drug development.

Figures

Figure 1

Inhibition of HIV-1 infection. 1G5 T cells were pretreated for 24 h with increasing concentrations of SP4-2, or with 10-6M ddC, or mock treated (0 μg SP4-2), as indicated. Then, cells were infected with HIV-1 (NL4-3) at multiplicity of infection (moi) of 0.01 for 1.5 h, washed 3 times, and returned to culture with the same concentration of each treatment, for the duration of the experiment. (A) On day 3 after infection, HIV-1 infection was quantified by luciferase gene marker expression from cell lysates that were normalized to the same number of viable cells, and expressed as relative light units (RLU) on the y-axis. (B) Viability for each cell culture treatment was quantified by MTT uptake. (C) Percent inhibition of HIV-1 was calculated from raw data in (A), utilizing the formula in the Methods, and plotted on the Y-axis as % HIV-1 Inhibition. Data are mean ± SD of three separate experiments.

Figure 2

Inhibition of X4 and R5-tropic HIV-1. GHOST X4/R5 and GFP expressing cells were plate at 1 × 105/well in 12-well plates and incubated at 37°C in CO2 atmosphere with increasing concentrations of SP4-2, as indicated, then infected with either X4-tropic NL4-3 (panel A, a-d) or with R5-tropic 81A (panel B, e-h), at 0.3 moi, in replicates (n = 4). 48 h after infection cells were quantified by FACS, and % infected cells is shown on each panel. Uninfected and untreated control (mock) is superimposed over each graph in dotted line. Representative of 4 experiments.

Figure 3

Inhibition of HIV-1 fusion. SupT1 cells (1 × 106) were (A) mock infected, (B) infected for 2 h at 0.5 moi with BlaM-Vpr-X4-tropic NL4-3, or (C) infected in the presence of 10 μg/ml SP4-2, or (D) infected in the presence of 250 nM AMD3100. In a parallel experiment, SupT1 cells (1 × 106) were either (E) mock infected, or (F) infected for 2 h at 0.5 moi with BlaM-Vpr-X4-tropic NL4-3, or (G) infected in the presence of 20 ng/ml sCD4, or (H) infected in the presence of 20 ng/ml sCD4 together with 16 μg/ml SP4-2. Cells were loaded with CCF2/AM dye and fusion was analyzed by multiparameter flow cytometry using a violet laser for excitation of CCF, and gated from 10,000 cells. Percentages in each panel are of cells displaying blue fluorescence (virus fusion positive cells). Representative of 3 separate experiments.

Figure 4

Inhibition of HIV-1 binding and replication. GHOST cells were plate at 1 × 105/well in 12-well plates and incubated at 37°C in CO2 atmosphere with increasing concentrations of SP4-2 for 1.5 hours prior to infection. Treatment was washed off 3 times with warm media and plates were transferred to 4°C for 2 h to cool. Then the cells were infected at 4°C with NL4-3 at 0.1 moi for 2 hours. (A) Unbound virus was removed by washing with cold PBS, and viral particles remaining bound to the cells were quantified by p24 ELISA. (B) In a parallel experiment, 4°C infected plates were returned to 37°C for 48 hours, and virus replication was quantified by p24 ELISA. Data are mean ± SD of 6 replicates.

Figure 5

Inhibition of post entry HIV-1 replication. (A) SupT1 cells were infected for 1.5 hours in the absence of any treatment, with HIV-1 chimera NL4-3 Env-Luc+/VSV-G pseudotype, washed 3 times, and then treated with increasing concentrations of SP4-2, for 24 h. Intracellular luciferase gene marker expression was quantified from cell lysates that were normalized to the same number of viable cells by the MTT assay, and percent inhibition of HIV-1 replication was calculated from a control cell culture of infected but untreated cells, and plotted on the y-axis. (B) Standard cell free fluorescent RT assay was performed in the presence of 2 units recombinant HIV-1 RT/reaction with the indicated concentrations of SP4-2. Percent inhibition was calculated comparative to assay performed in absence of treatment, 100% RT activity. Data are mean ± SD of three separate experiments.