?(Fig.1A).1A). 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. Background S. fusiforme is a species of brown Masitinib mesylate macroalgae (Class Phaeophyceae) that is commonly found in middle to lower rocky intertidal zones along the coastlines of China, Korea, Masitinib mesylate and Japan. Formerly called Hizikia fusiformis [1], it frequently occurs in dense aggregations. Individuals can be up to 1 1 m in length, with shorter side branches and narrow blades. It is frequently collected for human consumption. In our previous work with whole S. fusiforme extract, we reported up to 90% inhibition of HIV-1 Masitinib mesylate replication in several different cell types, including T cells and macrophages, both during entry and post-entry stages of the HIV-1 life cycle [2]. Importantly, this inhibition was also mediated against primary isolate R5-tropic HIV-1 (ADA) in human macrophages, and it also inhibited cell-to-cell fusion and subsequent viral spread to uninfected cells, which demonstrated ability of S. fusiforme Masitinib mesylate to inhibit physiologically relevant HIV-1 mechanism of infection. Based upon this work, we proposed that S. fusiforme mixture contained more than one biologically active molecule, and that it would be a lead candidate for bioactivity-guided isolation of active compounds mediating HIV-1 inhibition. Here, we report the isolation of a bioactive fraction SP4-2, with 230-fold enhanced antiretroviral activity against both X4 and R5-tropic HIV-1, specificity of inhibition of viral fusion mediated against CD4 receptor, and post entry inhibition of the HIV-1 RT. Compounds isolated from S. fusiforme have not been investigated until now [3,4]. Results Dose dependent inhibition of HIV-1 To begin characterization of the complex S. fusiforme extract, we performed bioactivity-guided fractionation, which resulted in identification of a biologically active fraction SP4-2 that we tested in T cells for the ability to inhibit HIV-1 Mouse monoclonal to FOXA2 infection (Fig. ?(Fig.1).1). Cells were treated with increasing concentrations of SP4-2, infected, and virus replication was measured by luciferase expression in 1G5 cells that were equalized to the same number of viable cells by the MTT assay (Fig. ?(Fig.1A).1A). Viability of treated cultures remained high and similar to that of mock and 10-6M ddC treated cells (Fig. ?(Fig.1B).1B). Maximal virus replication was determined from infected and untreated cells (0 g SP4-2), which expressed 29,601 luciferase relative light units (RLU), demonstrating active and ongoing virus replication (Fig. ?(Fig.1A).1A). Highly productive infection was confirmed by flow cytometry, with 99% of cells positive for HIV-1 antigens (data not shown). Comparatively, treatment with 2 g, 4 g, 6 g, and 8 g/ml SP4-2 reduced luciferase expression in a dose-dependent manner to 23,243, 13,253, 6,222, and 3,877 RLU, respectively. As expected, control cultures treated with 10-6M ddC, expressed background counts of 587 RLU, indicating almost total inhibition of virus replication (Fig. ?(Fig.1A).1A). We calculated percent HIV-1 inhibition in comparison to infected and untreated cells (Fig. ?(Fig.1C).1C). Treatment with SP4-2 inhibited virus replication in a dose dependent manner by 21, 55, 79, and 86%, respectively. The 50% inhibitory concentration (IC50) was calculated to be 3.7 g. Open in a separate window 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 g SP4-2), as indicated. Then, cells were infected with HIV-1 Masitinib mesylate (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.