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 infection (Fig. ). 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. ). Viability of treated cultures remained high and similar to that of mock and 10-6M ddC treated cells (Fig. ). Maximal virus replication was determined from infected and untreated cells (0 μg SP4-2), which expressed 29,601 luciferase relative light units (RLU), demonstrating active and ongoing virus replication (Fig. ). Highly productive infection was confirmed by flow cytometry, with 99% of cells positive for HIV-1 antigens (data not shown). Comparatively, treatment with 2 μg, 4 μg, 6 μg, and 8 μg/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. ). We calculated percent HIV-1 inhibition in comparison to infected and untreated cells (Fig. ). 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 μg.
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 (more ...)
S. fusiforme inhibits both X4 and R5-tropic HIV-1 infection
Next, we examined the cells coreceptor specificity and tested SP4-2 fraction for ability to inhibit both X4 and R5-tropic HIV-1 (Fig. ). GHOST cells expressing both X4 and R5 coreceptors were treated with increasing concentrations of SP4-2, and infected with X4-tropic NL4-3 (A) or with R5-tropic 81A (B), and FACS analyzed 48 h after infection. Treatment with SP4-2 resulted in a dose dependent decrease in number of infected cells by either virus. X4-tropic virus (A) infected 15.7% cells without treatment (a), which decreased to 13.5% (b), 7.6% (c), and 0.7% (d) infected cells after treatment with 1, 6, and 12 μg/ml SP4-2, respectively. Inhibition of infection was calculated to be 14%, 51%, and 95%, respectively. For R5-tropic infection, we observed a mean of 21% infected cells (e), which decreased to 19.9% (f), 17.5% (g), and 11.7% (h) infected cells after treatment with 1, 6, and 12 μg/ml SP4-2, respectively. Inhibition of infection was calculated to be 6%, 17%, and 45%, respectively. However, when we increased SP4-2 treatment to 14, 16, 20, and 24 μg/ml, R5 inhibition of infection increased proportionally to 65%, 70%, 78%, and 88%, respectively (not shown). Based on these results, we conclude that treatment with SP4-2 inhibits both X4 and R5-tropic HIV-1 infection in a dose dependent manner, confirming our previous results with whole S. fusiforme extract, which inhibited both X4 and primary R5-tropic HIV-1.
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 (more ...)
S. fusiforme inhibits HIV-1 fusion by blocking CD4 receptor
Viral entry into cells consists of two distinct steps of 1) virus binding to the cellular receptor and coreceptor, which is followed by 2) fusion of the viral and cellular membranes and internalization. To determine mechanism of the observed inhibition of infection, we tested for SP4-2 activity against HIV-1 fusion to CD4-expressing SupT1 T cells, by utilized a highly specific and sensitive fluorescence resonance energy transfer (FRET)-based HIV-1 fusion assay (Fig. ), [5
]. HIV-1 β-lactamase-Vpr (BlaM-Vpr) chimerical HIV-1 (NL4-3) was used to infect target cells that were loaded with CCF2/AM dye. Changes in CCF2 fluorescence reflect intracellular presence of BlaM, which is only present due to HIV-1 fusion and entry. Mock-treated negative control cells were loaded with dye, and were gated for background 520 nm emissions, which was low at 1.6% positive cells (0% fusion, panel A). After infection with BlaM-Vpr HIV-1, fusion was detected in 51.8% of the cells (100% fusion), as indicated by a shift to blue fluorescence (panel B). However, treatment of cells with 10 μg SP4-2 fraction inhibited this shift and markedly reduced viral entry, with only 25% of the cells being positive for viral fusion, which corresponded to 51.7% inhibition of the fusion (panel C). As a positive control for inhibition, we treated cells with 250 nM AMD3100 (CXCR4 inhibitor), which inhibited virus fusion, yielding 28.7% fusion positive cells that corresponded to 44.5% inhibition (panel D). Inhibition of fusion with AMD3100 increased to 80%, when we increased its concentration to 500 nM (not shown). From three different experiments we observed that treatment with 10 μg SP4-2 inhibited HIV-1 fusion by average of 53% (± 0.8 SEM).
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. (more ...)
Next, in a parallel experiment, we studied for the possible interaction between SP4-2 and CD4 (Fig. ). From 37% BlaM-Vpr HIV-1 fusion positive cells without any inhibitor (panel F), incubation with sCD4 only, resulted in 8.4% positive cells and blocked HIV-1 fusion by 77.2% (panel G). However, incubation of sCD4 together with SP4-2 resulted in 34% HIV-1 fusion positive cells (panel H), in effect reversing inhibition of fusion observed with sCD4 treatment. This result clearly indicates that SP4-2 interacts with CD4 receptor thereby blocking HIV-1 fusion to target cell.
S. fusiforme inhibits HIV-1 binding but not entry or replication
In addition to demonstrating inhibition of HIV-1 fusion by SP4-2-CD4 interaction, we were interested to define mechanism of this inhibition by investigating whether treatment with S. fusiforme prevents virus binding to the cell surface receptors in culture (Fig. ). Cells that are infected at 4°C allow only HIV-1 binding to the cell surface receptor but not fusion or entry. Except for 2 h SP4-2 pretreatment of cells that was done at 37°C to allow for SP4-2-CD4 interaction, we performed all the subsequent steps, including HIV-1 infection at 4°C. GHOST X4/R5 expressing cells were treated with increasing concentrations of SP4-2 (0–20 μg), and then washed three times with warm media to remove any unbound SP4-2. Next, cells were cooled and infected at 4°C with NL4-3 for 2 h, washed three times to remove any unbound virus, and bound HIV-1 was quantified from replicates (n = 6) by HIV-1 core antigen p24 ELISA (Fig. ). Treatment with 0, 12, 16, and 20 μg/ml SP4-2, resulted in a dose dependent decrease of HIV-1 bound to cells, which measured 860, 805, 435, and 331 pg/ml p24, respectively. The percent decrease in bound virus was calculated comparative to 100% bound virus (860 pg/ml p24), which was 6.3, 49.4, and 61.5%, respectively. Treatment with both 16 and 20 μg SP4-2 led to statistically significant decrease (p ≤ 0.0001) compared to no treatment (0 μg). To test whether HIV-1 bound at 4°C was capable of membrane fusion and replication, in a parallel experiment performed under same conditions, we returned the infected and washed cell cultures to 37°C for 48 h, and quantified virus replication by monitoring HIV-1 p24 production (Fig. ). Cell cultures pretreated with 0, 4, 8, 12, and 24 μg/ml SP4-2, replicated HIV-1 in a dose dependent manner that produced 1061, 807, 544, 352, and 148 p24 pg/ml, respectively. The HIV-1 inhibition was calculated to be 23.9, 48.7, 66.8, and 86%.
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 (more ...)
S. fusiforme inhibits HIV-1 reverse transcriptase
We showed that inhibition by whole S. fusiforme was mediated during several stages of the virus life cycle [2
]. To determine mechanism of this inhibition, we examined HIV-1 replication during post entry steps of the virus replication cycle (Fig. ). HIV-1 that is envelope deficient and is pseudotyped with VSV-G envelope bypasses any receptor entry restrictions and allows for a single round of infection, as previously demonstrated [7
]. To bypass inhibition at entry, we infected SupT1 cells with NL4-3 Env-
virus pseudotyped with VSV-G envelope for 2 h, and then added increasing concentrations of SP4-2 treatment. 24 h after infection, we measured luciferase production and calculated inhibition of virus replication in response to SP4-2 treatment (Fig. ). Treatment with 6, 10, and 12 μg SP4-2 inhibited post entry HIV-1 replication in a dose dependent manner by 50, 61, and 71%, respectively. Viability of treated cells, as quantified by MTT assay, remained similar to mock treatment (data not shown).
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 (more ...)
These data demonstrate that the HIV-1 is inhibited by SP4-2 after virus entry into cells. To examine the precise mechanism of the observed post entry inhibition, we investigated direct inhibition of recombinant HIV-1 RT, in a cell free assay. Treatment with increasing concentrations of SP4-2, with 0.078, 0.156, 0.313, 0.625, 0.125, and 2.5 μg, inhibited HIV-1 RT activity in a dose dependent manner by 4, 6, 17, 28, 47, and 79%, respectively (Fig. ). As a negative control for inhibition, we used a different fraction that was derived from whole S. fusiforme, which was shown to be inactive during bioactivity-guided fractionation. This fraction did not inhibit HIV-1 RT (not shown).