PPF appears to decrease viability of VSV-GFP infected trophoblasts and Jurkat cells (Fig. 1)
We conducted viability tests of trophoblasts, HeLa cells and Jurkat cells treated with varying concentrations of PPF from 0.001 to 10.0 μg/mL These cells were subsequently uninfected or infected with VSV (a prototypical enveloped virus that replicates in the cytoplasm of cells, similarly to ZIKV) fused to Green fluorescence protein (VSV-GFP) at a MOI of 1.0. PPF alone does not appear to greatly affect viability of trophoblasts, HeLa cells, or Jurkat cells. Infection with VSV-GFP reduces cell viability of all three cell lines. Increasing concentrations of PPF affects viability of infected trophoblasts. The most dramatic results are seen with HeLa cells, where cell viability drastically decreases in infected cells. Increasing the concentration of PPF does not further decrease viability of infected HeLa cells. In the case of infected Jurkat cells, increasing concentrations of PPF decreases the viability of infected cells.
PPF treated cells infected with VSV-GFP show formation of large extracellular vesicle (EV) filled with virus (Fig. 2)
Based on our hypothesis that PPF disrupts the membrane microenvironment of mammalian cells, thereby interfering with pathways of viral replication, we exposed Jurkat cells to PPF in vitro, at concentrations similar to those used for treatment of drinking water in Brazil. Control Jurkat cells, untreated with PPF and uninfected with VSV-GFP are shown in Fig. 2a. As expected, there is a lack of green fluorescence, a marker for the presence and replication level of virus (Fig. 2ai). Hoecsht staining reveals presence of nuclei, distinguishing cells (blue structures, Fig. 2aii) from vesicles, while differential interference contrast (DIC) microscopy (Fig. 2aiii) reveals normal morphology of cells. A merged image of i, ii and iii (Fig. 2aiv) shows Jurkat cells with normal morphology, stained nuclei and no virus.
Figure 2b represents cells untreated with PPF and infected with VSV-GFP. As expected, we see the presence of virus within cells, denoted by green fluorescence representing VSV-GFP (Fig. 2bi). Host cells supporting VSV-GFP replication eventually undergo apoptosis, as revealed by Fig. 2bii, which shows stained nuclei with a fragmented cell morphology that indicates initiation of apoptosis. DIC microscopy (Fig. 2biii) further confirms the apoptotic cell morphology. A merging of images i, ii and iii reveals the presence of VSV-GFP within nucleated cells (Fig. 2biv), again as expected. A few EVs were also observed, a phenotype that is normal for Jurkat cells (shown by white arrow in Fig. 2div) [20]. Some of the EVs showed the presence of virus, albeit very little, as reflected by the low intensity of green fluorescence measured by quantitative confocal microscopy (Fig. 3).
Our next step was to analyze Jurkat cells treated with PPF and uninfected with virus. As expected, there is a lack of green fluorescence (Fig. 2ci) and most cells appear to have normal morphology (Fig. 2cii, iii, iv). Analysis of PPF-treated and VSV-GFP infected cells revealed the presence of EVs packed with virus, as revealed by green fluorescence (Fig. 2di and iv), with the intensity of green fluorescence being proportional to the relative amount of viruses. The concentrated intensity of green fluorescence at the periphery of a large EVs shows high expression and localization of VSV-GFP at the membrane of the EV (Fig. 2di and v). The EV is a structure distinct from the Jurkat cell which is revealed by the presence of blue stained nucleus (yellow arrow, Fig. 2dii) adjacent to the EV. DIC microscopy (Fig. 2diii) shows the normal morphology of Jurkat cells (yellow arrow) and also depicts the shape of the large round EV devoid of a nucleus (red arrow). Jurkat cells appear smaller than the VSV-filled EV (Fig. 2diii, red arrow) and the two structures seem interconnected by a tubule (pink arrow). VSV-GFP packed EV vary in size and shape. Some of these structures appear attached to cells (pink arrows, Figs. 2diii and dviii) while others are free standing. Figure 2dv-viii represent a different field of view and show what appear to be vesicles of various sizes and shapes, seemingly emerging from cells (red arrow, Fig. 2dvii), connected by tubules (pink arrows). Figure 2dviii shows more intense green fluorescence (corresponding to more viruses) in attached EVs than in surrounding Jurkat cells (yellow arrow).
The variety and number of EVs suggests their presence to be an active process needing the use of mitochondria, the energy powerhouse of the cell. Our next step was to investigate the presence of active mitochondria in these structures, knowing that VSV interacts/co-localizes with mitochondria [21, 22].
PPF treated cells infected with VSV-GFP accumulate viruses as well as mitochondria in extracellular vesicles (EVs) (Fig. 3)
VSV is known to interfere with mitochondria, diverting cellular energy metabolism towards enhancing viral replication and ultimately inducing cell apoptosis [22]. We subjected Jurkat cells treated or untreated with PPF with or without VSV-GFP infection, to quantitative confocal microscopy. We assessed the fluorescence intensity of active mitochondria (orange channel corresponding to Mito-tracker Orange), VSV-GFP (green channel) and nuclei (blue channel), with cell membranes (black graph) representing differential interference contrast. Figure 3a shows background fluorescence levels of Jurkat cells untreated with PPF and uninfected with VSV-GFP. The inset shows a few cells, stained blue for nuclei and orange for mitochondria, with the profiled fluorescence pattern representing the cell highlighted within the red box. As expected, there is a lack of green fluorescence. Orange fluorescence represents an uneventful state for the mitochondria. The peak in blue fluorescence represents nuclei of healthy cells, while the black graph represents structural integrity of the cell membrane.
Figure 3b and c both show PPF treated cells uninfected with virus, with the fluorescence profile representing the cell highlighted by the red box. The fluorescence profile seen in 3B is different from that seen in 3A and is likely an effect of PPF on the individual Jurkat cells. Figure 3c shows disturbed cell membrane (black graph) suggestive of apoptotic cell morphology (see inset), while Fig. 3b profiles an intact cell. Fluorescence analysis of cells untreated with PPF and infected with VSV-GFP showed cells filled with virus. When a large, green cell representing a Jurkat cell with VSV-GFP was analyzed by quantitative confocal microscopy (Fig. 3d), we saw viruses (green channel) co-localizing with nuclei (blue channel) and also with mitochondria (orange channel). Mitochondria (Fig. 3d) showed normal fluorescence, similar to the orange fluorescence profiles in Fig. 3a, b and c, implying background levels of engagement during VSV-GFP replication in Jurkat cells untreated with PPF.
PPF treated cells infected with VSV-GFP showed presence of virus within cells and EVs (Fig. 3e and f). Fluorescence analysis of a large EV (see inset, Fig. 3e) that appears attached to a cell, reveals presence of viruses (green channel), mitochondria (orange channel), and cell membrane (black graph), but no nuclear staining (blue channel), since the EVs are devoid of nuclei. The distribution of VSV-GFP inside these large EVs is very unusual, showing pockets of virus accumulated inside the vesicles as well as at the periphery or vesicle surface (quantitative analysis of Fig. 2di). The accumulated virus pockets within the Jurkat cells are also rich in mitochondria, with which the virus is co-localizing. Figure 3f shows the fluorescence profile of the cell (shown within the insert) attached to the large EV that was analyzed in Fig. 3e. Fluorescence analysis of this cell, treated with PPF and infected with virus reveals co-localization of nuclei (blue channel) and mitochondria (orange channel), with green fluorescence above the background levels seen in cells uninfected with virus (Figs. 3a, b and c). Higher green fluorescence in EVs (Fig. 3e) suggests higher viral load compared to cells untreated with PPF (Fig. 3d).