S. fusiforme does not inhibit cell growth or viability
To establish a non-toxic working concentration, we tested for cell growth and viability kinetics in response to treatment with S. fusiforme whole aqueous extract. T cells were treated with either 2 or 4 mg/ml S. fusiforme, 10-6 M ddC, or were mock treated (Fig ). In 1G5 cells, growth kinetics remained similar, except for the highest 4 mg/ml treatment on day 7 that decreased cell growth by 19% compared to ddC treatment, indicating possible toxicity at this dose (Fig ). In parallel we also measured cell viability by trypan blue exclusion assay. Regardless of treatment, cell viability remained above 90%, which was comparable to mock treated cultures (Fig. ). We repeated this experiment with HIV-1 infected 1G5 cells, with similar results (not shown). Because of toxicity relevance in primary human cells, we also measured cell growth and viability in human peripheral blood mononuclear cells (PBMC), with similar results (Fig and ). Cells treated with either 3 or 4.5 mg/ml S. fusiforme exhibited somewhat slower growth kinetics on day 6 after treatment, as compared to 1.5 mg/ml S. fusiforme, ddC or mock treated cells (Fig ). However, viability of S. fusiforme and ddC treated cells remained similar through day 6 of follow-up, with the overall PBMC's viability declining over time, as compared to 1G5 T cell line (compare Fig. to ).
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 (more ...)
Based on these results we conclude that treatment with less than 4 mg/ml S. fusiforme extract, does not inhibit cell growth, is not toxic to cells, and is suitable for in vitro testing of HIV-1 inhibition in 1G5 cells.
S. fusiforme inhibits HIV-1 infection in T cells in a dose dependant manner
Next, we investigated S. fusiforme
ability to inhibit HIV-1 infection in T cells. We chose 1G5 T cells, which are stably transfected with HIV-LTR-luciferase gene construct, have low basal level of luciferase expression and are sensitive to HIV-1 tat
activation, which makes them a useful tool for testing HIV-1 inhibitors [28
]. Cells were treated with increasing concentrations of S. fusiforme
extract and infected with NL4-3. On day 3 after infection, equal numbers of viable cells were analyzed for intracellular luciferase expression, and cell viability was measured by MTT uptake assay (Fig. ). Percent HIV-1 inhibition was calculated by comparison to control infected untreated cell cultures, which expressed 18,797 relative light units (RLU) of luciferase (not shown). Treatment with 1.5, 3, and 6 mg/ml of S. fusiforme
extract inhibited HIV-1 replication in a dose dependant manner, by 60.4, 86.7, and 92.3%, respectively (Fig. ). As expected, treatment with positive control HIV-1 reverse transcriptase (RT) inhibitor ddC, blocked virus replication by over 98% (not shown). In parallel, we tested for the MTT uptake by viable cells, which remained high regardless of S. fusiforme
treatment, and was similar to ddC, as well as to viability of mock treated cells (Fig. ).
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) (more ...)
Based on these results we conclude that S. fusiforme treatment inhibits HIV-1 replication in T cells in a dose dependant manner, inhibition is similar to that achieved with ddC treatment, and treatment is not toxic to cells.
S. fusiforme inhibition is non-toxic and can be sustained over extended periods
Next, we tested for the duration of HIV-1 inhibition in 1G5 T cells, treated with either 2 mg/ml S. fusiforme or with 10-6 M ddC. Infection was monitored by luciferase expression from cells equalized to same number of viable cells by MTT assay, at the indicated time points after infection (Fig. ). HIV-1 infection in untreated cells gradually increased from 16,110 RLU expressed on day 3, to 86,720 RLU on day 7 after infection, which demonstrated highly productive and de novo HIV-1 synthesis (not shown). Treatment with 2 mg/ml S. fusiforme inhibited this infection by 77, 99, and 99% on day 3, 5, and 7, respectively (Fig. ). As expected, inhibition by ddC was 99% at each time point tested. Based on these results we calculated IC50 to be 0.86 mg. Similar time course inhibition results were obtained in CEM T cells (not shown).
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 (more ...)
In parallel to infection kinetics, we also tested cell viability by trypan blue exclusion assay (Fig. ). Cell viability in S. fusiforme treated cultures remained high at 98, 94, and 97% viable cells on day 3, 5, and 7, respectively. Cell viability in ddC treated cultures was similar, and measured 94, 93, and 97% viable cells on day 3, 5, and 7, which was similar to mock treated cultures. This data confirm MTT viability results, which were used to equalize cells to same numbers of viable cells (not shown).
Collectively, these findings demonstrate that S. fusiforme inhibits infection and de novo HIV-1 synthesis, through day 7 of follow-up, and this treatment does not affect cell viability.
S. fusiforme blocks HIV-1 transmission by direct cell-to-cell mechanisms of infection
HIV-1 infection is spread either by free viral particles, or 100 times more efficiently by direct cell-to-cell fusion [1
]. Considering that S. fusiforme
inhibits HIV-1 infection in T cells (Fig. ), we wanted to determine its ability to block cell-to-cell mediated viral transfer. To test this, we performed two separate experiments with different cell types (Fig ). First, we examined the ability of HIV infected CEM cells to fuse and spread infection to uninfected 1G5 cells that were either mock treated, treated with 10-6
M ddC only, or treated with increasing concentrations of S. fusiforme
and ddC, or with S. fusiforme
only. Pretreatment of 1G5 cells with 10-6
M ddC inhibits virus replication, and therefore serves as a control for false positive luciferase readings from free virus particle infection and replication, however it does not prevent spread of infection by cell-to-cell fusion. CEM and 1G5 cells were cocultivated for 24 h at a ratio of 1:1, and examined for cell-to-cell fusion and syncytia formation by phase contrast microcopy (A-F) or by luciferase expression (H). As expected, many large syncytia were observed in co-cultures with mock treated or only ddC treated 1G5 cells (A and B). However, 1G5 treatment with 2 mg S. fusiforme
, with or without ddC, greatly reduced cell-to cell fusion and syncytia formation (C and E). No giant cells were detected in 1G5 cells treated with either 4 mg/ml (D and F) or with 6 mg/ml (not shown) S. fusiforme
, with or without addition of ddC. Inhibition of viral infection by cell-to-cell fusion was also confirmed by decreased luciferase expression in S. fusiforme
treated 1G5 cells that were cocultivated with HIV infected CEM cells (H). CEM cells do not have the HIV-LTR-luciferase gene, as 1G5 cells do, and therefore luciferase readings from cocultivated cell cultures can only arise from 1G5 cells that fused and formed giant cells with infected CEM cells. 24 h after cocultivation with untreated 1G5 cells, luciferase expression measured 1.9 × 105
RLU, which represented maximal luciferase expression in the absence of any treatment (not shown). 1G5 treatment with 10-6
M ddC and 2, 4, or 6 mg S. fusiforme
inhibited cell-to-cell fusion, as measured by luciferase expression in 1G5 cells, by 77, 96, and 98%, respectively (H). Inhibition was similar in cells treated with S. fusiforme
only, in the absence of ddC, demonstrating low rate of infection by free virus, during the 24 hours of cocultivation (not shown). In comparison, 1G5 cell treatment with only 10-6
M ddC, inhibited luciferase expression by 69%.
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) (more ...)
In the second experiment, we cocultivated HIV infected and untreated 1G5 cells with uninfected and treated HIV-LTR-GFP-expressing GHOST adherent cells [29
], and monitored for cell-to-cell fusion by GFP expression from GHOST cells (G). After cocultivation with infected 1G5 cells, mock or only ddC treated GHOST cells can fuse, and form syncytia that emit green florescence, which was detected by phase fluorescence microscopy. GHOST cells that were ddC treated and cocultivated with HIV-1 infected 1G5 cells, resulted in cell-to-cell fusion and fluorescent giant cell formation as is shown by fluorescence micrograph superimposed on the phase contrast black and white image of the same field (G). However, as in CEM-1G5 cocultivation experiment, no giant cells emitting green fluorescence were detected in 1G5 cells cocultivated with GHOST cells that were treated with S. fusiforme
, with or without ddC (not shown).
Based on the results of these two different experiments, we conclude that S. fusiforme blocks HIV-1 infection by cell-to-cell fusion mechanism, which also prevents subsequent multinucleated cell formation and its associated cytophatic effects.
S. fusiforme inhibits HIV-1 infection in primary human macrophages and brain microglia
Macrophages and brain microglia are productively infected with R5-tropic HIV-1, and are considered to be the primary source of virus replication in the periphery and in the CNS [1
]. Because of their importance to HIV infection, we investigated ability of S. fusiforme
extract to inhibit virus infection in these cells. Primary human macrophages or microglial cell cultures were treated with 1 mg/ml S. fusiforme
extract and infected with primary R5 isolate ADA [30
]. Infection was monitored by measuring viral p24 concentrations in cell-free supernatants, at the indicated time points after infection (Fig. ).
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 (more ...)
In infected and untreated macrophage cell cultures, virus levels steadily increased from 19,097 pg of p24/ml on day 4, to a peak of infection on day 14, measuring 163,740 pg of p24/ml, indicating productive HIV-1 infection and de novo virus synthesis (not shown). However, treatment with 1 mg/ml S. fusiforme extract inhibited ADA replication (dark bars) by over 90% through day 14 after infection, which was comparable to the inhibition with ddC treatment (Fig. ).
Next, we treated fetal microglial cell cultures with either 1 mg/ml S. fusiforme, or 10-6 M ddC, or mock treated, and monitored infection kinetics by p24 production in cell-free supernatants at the indicated time points after infection (Fig. ). As in T cells and macrophages, infected and mock treated microglia were productively infected as demonstrated by steadily increasing p24 production that reached a peak on day 14 with 2,313 pg of p24/ml (not shown). Treatment with S. fusiforme inhibited this infection by 75% on day 3, by over 90% on day 7 and 10, and by 81% on day 14 after infection. By comparison, virus inhibition by ddC was 72% on day 3, and thereafter remained above 90%.
In parallel to infection kinetics, we monitored cell viability by MTT assay, which remained high and was similar to uninfected cell cultures (not shown). Based on these results we conclude that S. fusiforme is a potent inhibitor of R5-tropic HIV-1 infection in primary human macrophages and microglia: inhibition is long lasting, not toxic to cells, and with similar inhibition kinetics to those observed in T cells (Fig. ).
S. fusiforme inhibits HIV-1 infection during entry and post-entry events of virus life cycle
Collectively, our results demonstrate that S. fusiforme extract robustly inhibits HIV-1 infection in a number of cell types, and in a number of infection scenarios. In order to determine how this inhibition works, we tested whether the extract could block infection at a post-entry level of virus replication.
HIV-1 pseudotyped with the vesicular stomatitis virus G-protein (VSV-G) can infect cells without interacting with CD4 and co-receptors. We extended HIV-1 tropism by pseudotyping native HIV-1 (NL4-3) with VSV-G envelope (VSV/NL4-3), which produced native NL4-3 with heterologous envelope glycoproteins that bind to commonly expressed cellular receptors. VSV/NL4-3 virus gains access to the cytoplasm by fusing out of endocytic vesicles [31
]. Therefore, any block to VSV/NL4-3 replication would suggest post-entry inhibition. We treated T cells with increasing doses of S. fusiforme
, infected with NL4-3 or VSV/NL4-3, and monitored infection by luciferase gene expression on day 3 after infection (Fig. ). To our surprise, S. fusiforme
mediated dose dependant inhibition of VSV/NL4-3, inhibiting at 26.6, 32.8, and 62.6% that corresponded to 1, 2, and 3 mg/ml S. fusiforme
extract treatment, respectively (Fig. , light bars). However, overall inhibition of pseudotyped virus was markedly lower as compared to inhibition of native NL4-3, which was inhibited by 53, 78, and 93% (dark bars). Considering that pseudotyped VSV/NL4-3 has no cell surface entry restrictions, these data suggest that: 1) S. fusiforme
blocks at a post-entry step of viral replication, and 2) inhibition is also mediated during entry process, as suggested by difference in the levels of inhibition between native NL4-3 and VSV/NL4-3 infections.
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, (more ...)
To confirm and extend the finding of post-entry inhibition in T cells, we tested for inhibition of VSV/NL4-3 in CD4-negative primary cells. Human astrocytes are CD4-negative cells that are nonproductively infected by HIV-1 in vivo
], and in vitro
]. However, we showed that, in vitro
, these cells fully support productive virus replication after entry restriction has been bypassed [35
]. Infection with VSV/NL4-3 productively infects majority of astrocytes, and serves as model system to study HIV-1 replication in these cells [35
]. We infected primary human astrocytes with VSV/NL4-3, and monitored infection kinetics at the indicated time points after infection, by measuring p24 production in cell-free culture supernatants (Fig. ). Peak of infection was reached on day 12 with 71,000 pg of p24/ml produced in the infected and untreated cell culture, indicating ongoing virus replication (data not shown). Consistent with post-entry inhibition observed in T cells (Fig. ), treatment with 1 mg/ml S. fusiforme
extract also inhibited post-entry virus replication in primary human astrocytes, by 71, 40, and 54%, on day 3, 6, and 12, respectively (Fig. ).
These data support our hypothesis that in addition to inhibiting viral entry, S. fusiforme extract also blocks viral replication during a post-entry event of the virus life cycle. However, the exact mechanisms of either entry or post-entry inhibition need to be further investigated.