CEM, Jurkat and U937 cells were obtained from ATCC (Manassas, VA, United States) and maintained in RPMI-1640 media containing 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine, and 1% streptomycin/penicillin (Quality Biological, Gaithersburg, MD, United States). Monocytic U937 cells were induced to differentiate into monocyte-derived macrophages (MDM) with 50 nM of phorbol-12-myristate-13-acetate (PMA; Sigma–Aldrich) for 5 days. The primary monocytes were isolated from peripheral blood mononuclear cells (PBMCs; purchased from Precision Biosciences) and maintained in RPMI-1640 media as described above. Primary monocytes isolated from PBMCs were induced to differentiate into MDMs with 10 nM of phorbol-12-myristate-13-acetate (PMA) for 2 days. HEK-293T cells were maintained in Dulbecco’s modified minimum essential medium (DMEM) media containing 10% heat-inactivated FBS, 1% L-glutamine, and 1% streptomycin/penicillin (Quality Biological, Gaithersburg, MD, United States). All cells were incubated at 37°C in the presence of 5% CO₂.

For antibiotic selection of transfected 293T cells, Hygromycin B (Invitrogen), Zeocin (Life Technologies), or Geneticin (Life Technologies) were added to the culture next day. Hydroxyurea (Sigma–Aldrich) and r-Roscovitine (Cell Signaling Technology) were used for the treatment of transfected cell cultures for in vivo labeling followed by kinase assay. Other Cdk2 inhibitors used for kinase assays (Alsterpaullone, Indirubin-3′-monoxime, and Purvalanol A) were purchased from Sigma–Aldrich. Treatment of transfected 293T cells with Oxytetracycline (Selleck Chemicals), Esomeprazole (Selleck Chemicals), and Cambinol (Sigma–Aldrich) for analysis of levels of exosomal markers took place the day following transfection. All experiments involving biohazards were carried out under the IBC-approved institutional biosafety guidelines and were performed at BSL-2 level.

Ebola structural proteins were expressed from plasmids (Invitrogen) with CMV promoters and specific antibiotic selection markers: GP (pcDNA3.1/Zeo), NP [pcDNA3.1 (±)], VP40 (pcDNA3.1/Hygro). Twenty microgram of Escherichia coli-purified DNA was transfected into 293T cells using either Attractene reagent (Qiagen, Chatsworth, CA, United States) according to the manufacturer’s instructions, or by electroporation as previously described (Kashanchi et al., 1992). Transfected cells were treated next day with either 100 μg/mL Zeocin (CMV-GP), 500 μg/mL Geneticin (CMV-NP), or 200 μg/mL Hygromycin B (CMV-VP40) for plasmid selection. To generate resistant clones, transfected cells were cultured for >2 weeks, followed by isolation of surviving colonies (<1% of cells) and multiple passages under specific antibiotic selection. A total of 13 resistant clones were generated, but only 2 survived after >10 passages: one GP-clone (data not shown) and one VP40-clone (EVTR2C). All resistant clones were kept under constant antibiotic selection.

The nanotraps utilized here are multifunctional hydrogel particles 700–800 nm in diameter composed of high affinity aromatic baits containing a core surrounded by a sieving shell with pores that will selectively allow the passage of smaller molecules. Polymerization reactions with reactants N-isopropyl acrylamide (NIPAm), N,N-Methylenebisacrylamide (Bis) and either allylamine (AA), acrylic acid (AAc) or methacrylate (MA) are required for the synthesis of the particle’s shell. Vinyl sulfonic acid (VSA) monomers may also be incorporated into the shell for greater exclusion of high molecular weight and highly abundant analytes. Specific high affinity charge-based baits are covalently attached to the core matrix and bind target molecules through hydrophobic and electrostatic interactions, preventing degradation while sequestering the targets. NT80 beads contain Reactive Red 120 core bait while NT82 beads have Cibacron Blue F3GA. Both bead types have a NIPAm-Bis-AA matrix with a shell lacking VSA (Jaworski et al., 2014b). The characteristics of the newer nanotrap particles utilized in this manuscript are outlined in Figure 3B. For the capture and isolation of exosomes from transfected cell culture supernatants, a 20 μL slurry (30%) of 1:1 NT80 and NT82 nanotrap particles (Ceres Nanosciences) was incubated with 500 μL of cell-free supernatant. For capture of exosomes from resistant clone cell culture supernatants, a 25 μL slurry (30%) of NT80/82 beads (1:1) was incubated with 1 mL of cell-free, filtered (0.22 micron) supernatant. Samples were bound at 4°C for 72 h. NT pellets were isolated and washed with 500 μL of sterile 1x PBS without calcium and magnesium, followed by resuspension in 20 μL of Laemmli buffer for SDS/PAGE. For the capture and isolation of VP40, 69 ng of E. coli-purified VP40 was spiked into 100 μL of PBS and incubated with 20 μL of slurry (30%) of nine different NTs (210, 212, 213, 217, 219, 222, 223, 224, and 229) overnight at 4°C. Beads were washed with sterile 1x PBS without calcium and magnesium and resuspended in 20 μL Laemmli buffer for SDS/PAGE. For the capture and isolation of VLP in the presence of SDS, 20 μL of 30% slurry of the 9 NTs listed above was incubated with 1 μg of purified VLP in 80 μL SDS buffer [1:4; 20 μL Laemmli buffer and 60 μL Radioimmunoprecipitation assay (RIPA) buffer] overnight at 4°C. NT pellets were washed once with 500 μL of TNE50 + 0.1% NP40, followed by resuspension in Laemmli buffer for SDS/PAGE. For capture and isolation of VLP-derived VP40 in human samples, 100 μL of healthy human saliva (BioreclamationIVT, Baltimore, MD, United States), urine (obtained from a healthy male lab worker), and serum (#AY217; NCI bank transferred to FK) from healthy anonymous donors were diluted 1:3 in sterile 1x PBS without calcium and magnesium and spiked with 2.5 μg of purified Ebola VLP. One hundred and fifty microliters each of RIPA and Laemmli buffer (total 300 μL of 1:1 SDS buffer) was added to each sample, followed by addition of 60 μL of a 30% slurry of NT219. Samples were incubated overnight at 4°C, washed once with 500 μL of TNE50 + 0.1% NP40, and resuspended in 20 μL of Laemmli buffer for SDS/PAGE.

Cells were seeded into 96 well plates at 50,000 cells per well in fresh media followed by treatment. For treatment of CEM, Jurkat, and U937 cells with VLP or VP40, 0.1 or 0.5 μg of VLP or E. coli-purified VP40 (generously provided by IBT Bioservices) was placed into 100 μL of cells suspended in fresh media. For treatments with cell culture supernatants, transfected cells or resistant clones were cultured for 5 days to maximize the concentration of exosomes within the media as previously described (Narayanan et al., 2013). Media was then harvested from the cells, centrifuged for 10 min at 14,000 rpm to remove cells and cellular debris, and either filtered (0.22 micron) or left unfiltered. Seeded cells in fresh media (50 μL total) were then treated with 50 μL of supernatant. All treatments of CEM, Jurkat, and U937 cells were allowed to incubate for 5 days followed by measurement of cell viability using CellTiter-Glo Cell Luminescence Viability kit (Promega, Madison, WI, United States) as per manufacturer’s instructions. Briefly, 100 μL CellTiter-Glo reagent was added to the wells (1:1 reagent:cell suspension) and incubated at room temperature for 10 min followed by detection of luminescence using the GLOMAX multidetection system (Promega). Primary macrophages were assayed for cell viability 2 days following treatment.

Cell pellets were harvested, washed twice with 1x PBS without calcium and magnesium, and resuspended in lysis buffer [50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 50 mM NaF, 0.2 mM Na₃VO₄, 1 mM DTT, and 1 complete protease inhibitor mixture table/50 mL (Roche Applied Science, Mannheim, Germany)]. The suspension was incubated on ice for 20 min with gentle vortexing every 5 min, followed by centrifugation at 10,000 rpm at 4°C for 10 min. Protein concentration from the lysate supernatant was determined using the Bradford protein assay (Bio-Rad).

Whole cell extracts (10 μg) were resuspended in 10 μL Laemmli buffer, heated at 95°C for 3 min, and loaded onto a 4–20% Tris-glycine SDS gel. Nanotrap particle pellets were resuspended in 15 μL Laemmli buffer, heated at 95°C for 3 min and vortexed three times until fully resuspended, and then loaded onto a 4–20% Tris-glycine SDS gel. Gels were run at a maximum 160 V and transferred overnight onto PVDF membranes. Membranes were blocked in 5% milk in 1x PBS containing 0.1% Tween 20 for 1 h at 4°C, then incubated overnight at 4°C with appropriate primary antibody: [α-Caspase 3, α-PARP-1, α-Alix, α-CHMP6, α-TSG101, α-EAP20, α-EAP45, α-TRBP (Santa Cruz Biotechnology); α-Dicer, α-Drosha, α-Ago1, α-Exportin 5, α-DGCR8, α-CD63, α-β-actin, (Abcam); α-VP40 and α-GP] were generously provided by IBT Bioservices. Membranes were then incubated with the appropriate HRP-conjugated secondary antibody for 2 h at 4°C and developed using SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL, United States). Luminescence was visualized on a Kodak 1D image station (Carestream Health, Rochester, NY, United States). Raw densitometry counts were obtained using ImageJ software.

Mass spectra for VP40 was obtained for this manuscript as previously described in our analysis of VP30 from EBOV virions (Ilinykh et al., 2014). Proteomics data for VP40 had not been previously published. The spectra were analyzed by SEQUEST (Thermo) search engine. Protein phosphorylation sites were detected with Proteome Discoverer 1.4 software and mapped to the VP40 protein sequence.

Immunoprecipitation (IP) was performed by incubation of 500 μg of CEM or transfected and treated 293T whole cell extracts with 10 μg of appropriate primary antibody (α-Cdk2, α-CycE, α-CycA, α-normal rabbit IgG; Santa Cruz Biotechnology) and 100 μL TNE50 + 0.1% NP-40 for 48 h at 4°C. CEM cells were utilized for these experiments as we have previously shown that these cells contain active Cdk/Cyclin complexes that can easily be purified using specific antibodies (Wang et al., 2001). The next day, complexes were precipitated with 30 μL of a 30% slurry of A/G beads (Calbiochem) for 2 h at 4°C, washed twice with TNE50 + 0.1% NP-40 and twice with kinase buffer. The reaction mixtures (20–30 μL) contained the following final concentrations: 40 mM β-glycerophosphate (pH 7.4), 7.5 mM MgCl₂, 7.5mM EGTA, 5% glycerol, [γ-³²P] ATP (0.4 mM, 1 μCi), 50 mM NaF, 1 mM orthovanadate, and 0.1% (v/v) β-mercaptoethanol. Phosphorylation reactions were performed with immunoprecipitated material and E. coli-purified VP40 (0.5 μg) as a substrate in TTK kinase buffer containing 50 mM HEPES (pH 7.9), 10 mM MgCl₂, 6 mM EGTA, and 2.5 mM dithiothreitol. Reactions were incubated at 37°C for 1 h, stopped by the addition of 1 volume of Laemmli sample buffer containing 5% β-mercaptoethanol, and run on 4–20% SDS-polyacrylamide gel. Gels were subjected to autoradiography and quantification using PhosphorImager software (Amersham Biosciences). Kinase assay with peptides was accomplished by adding 50 μg appropriate peptide [S3 (ADLTSPEKIQAI), S3M (ADLTAPEKIQAI), T7 (GKKVTAKNGQPI), S8 (GKKVASKNGQPI), T2 (LVHKLTGKKV) (Biomatik USA, LLC, Wilmington, DE, United States)] and 15 μL of appropriate antibody (α-CycE, α-IgG) to the Protein A/G pellet suspended in kinase buffer (120 μL). Next, [γ-³²P] ATP was added to the samples, incubated at 37°C for 1 h, and samples (10 μL) were dotted onto Whatman glass microfiber filters, dried for 30 min, and subsequently submerged in TE buffer with gentle agitation for 48 h. Samples were quantified using a scintillation counter (QuantaSmart^TM DPM assay count).

For in vivo labeling, 293T cells (5 × 10⁶) were electroporated with 20 μg of VP40 plasmid, followed by addition of Hygromycin B (200 μg/mL). Cells were grown up to 30–40% confluency (4 days) at which time Hydroxyurea (G1/S blocker; 1 mM) was added for one additional day. Media were removed and 1 mL of DMEM was added to cover the cells, with the addition of 10 μL of [γ-³²P] ATP (∼3000 mCi/mL) for 4 h. Next, r-Roscovitine (1–10 μM) was also added to a few of the samples. After labeling, cells were chased with cold complete media (no radioactivity) for 2 h. Cells were removed with a cell scraper and lysed in lysis buffer, followed by IP with α-VP40 antibody overnight in TNE150 + 0.1% NP-40. Protein A/G was added and bound beads were washed 2x with TNE150 + 0.1% NP-40 and once with kinase buffer. Pellets were then resuspended in Laemmli buffer and run on 4–20% SDS-polyacrylamide gel. Gels were subjected to autoradiography and quantification using PhosphorImager software (Amersham Biosciences).

293T, transfected 293T, and EVTR2C cells were grown in DMEM containing 10% heat-inactivated FBS, 1% L-glutamine, and 1% streptomycin/penicillin (Quality Biological, Gaithersburg, MD, United States). Exosome preparation was made from 100 mL of cell culture supernatant (produced from fully confluent cells grown for 5 days). Cells were pelleted by centrifugation at 300 g for 10 min. An additional centrifugation at 2000 g for 10 min was used to pellet dead cells. The resulting supernatant was then filtered through a 0.22 micron filter and ultracentrifuged at 10,000 g for 30 min. This resulting supernatant was then pulled off and ultracentrifuged at 100,000 g for 70 min to pellet the exosomes. These exosomes were then washed with 22.5 mL of sterile 1x PBS without calcium and magnesium and ultracentrifuged again at 100,000 g for 70 min. The supernatant was pulled off and the final pellet was resuspended in 100 μL of PBS. All spins were performed at 4°C. Protein levels of the exosomes were determined with the Amplex^® Acetylcholine/Acetylcholine Esterase Activity Assay Kit (Thermo A12217) following the manufacturer’s instructions. Briefly, a 1x running buffer negative control (20 mL of H₂O and 5 mL of 5x reaction buffer) and two positive controls, one consisting of acetylcholine esterase and one consisting of hydrogen peroxide, were made and plated on a 96-well plate. Exosomes were treated and fluorescence of acetylcholine esterase activity was measured with a GLOMAX multidetection system (Promega) every 15 min for 1 h to find optimal activity.

Standard deviation was calculated in all quantitative experiments done in triplicate. All p-values were calculated using two-tailed Student’s t-tests (Microsoft Excel) and were considered to be statistically significant when p < 0.05.

It has been well documented that EBOV as well as Ebola VLPs are capable of inducing significant cell death as well as cytokine storm in vitro and in vivo (Wauquier et al., 2010; Wahl-Jensen et al., 2011; Falasca et al., 2015; Messaoudi and Basler, 2015), particularly in macrophages and dendritic cells. Additionally, bystander lymphocyte apoptosis has been observed in many experimental and clinical models of Ebola infection (Geisbert et al., 2003; Wauquier et al., 2010). However, direct cell regulation of lymphocytes has not been studied by Ebola viral VLPs or structural proteins. We hypothesized that one or more of EBOV proteins may cause deregulation of cell growth on recipient immune cells. To this end, we incubated increasing concentrations of VLP and VP40 with three immune cell types, including two types of T-cells (early and late developmentally deregulated cells represented by CEM and Jurkat cells, respectively) and one monocyte cell line (U937), and assayed for cell viability using CellTiter-Glo. Purified E. coli-made VP40 was utilized, as the other two proteins GP and NP also found in VLPs were insoluble using prokaryotic vectors (data not shown). Only two concentrations of VLP and VP40 were used for these experiments due to limited availability of reagents. Results in Figure 1A show that VLPs (at varying concentrations) were not apoptotic in lymphocytes, while monocytes showed a slight decrease in cell viability at higher VLP concentrations. On the other hand, when using E. coli-purified VP40, the two T-cell lines showed a decrease in viability (Figure 1B). To verify the relative levels of VP40 in the purified VP40 versus that found in the VLPs, assessment by Western blot showed a slightly higher amount of VP40 (approximately 1.75-fold higher levels of VP40 compared to VLP, as determined by densitometry analysis) in the E. coli-purified preparation than that in VLPs (Figure 1C). Importantly, VP40 within VLPs is likely to be hidden under the envelope, therefore only the unmasked VP40 decreased T-cell viability shown in Figure 1B. We have recently shown that analysis of apoptotic markers Caspase 3 and PARP-1 by Western blot in the case of Rift Valley Fever virus infection indicated cell death by apoptosis in Jurkat cells treated with exosomes containing proteins from RVFV resistant clones (Ahsan et al., 2016). Similarly, here T-cells and monocytes treated with VP40 showed increased pro-Caspase cleavage and accumulation of cleaved PARP-1 (Figure 1D), confirming that cells treated with free VP40 protein initiated programmed cell death. The levels of Actin in the VP40-treated lanes (lanes 3, 6, and 9) decrease as well, which is also indicative of cell death. Collectively, these results indicate that free VP40 can induce apoptosis in immune cells, especially in T-cells.

Ebola-infected cells are capable of production and release or accumulation of EBOV proteins in a cell type dependent manner (Steele et al., 2001). As we have previously shown dysregulation of immune cells by supernatants from HIV, HTLV, and Rift Valley Fever virus infected cells (Narayanan et al., 2013; Jaworski et al., 2014a; Ahsan et al., 2016; Sampey et al., 2016), we wished to determine whether supernatants from cells transfected with Ebola structural protein-containing plasmids would impact the viability of neighboring uninfected cells. We transfected 293T cells with VP40, GP, and NP plasmids followed by specific antibiotic selection for each plasmid. The supernatants from the transfected cells were isolated and used to treat uninfected CEM, Jurkat, and U937 cells. Cell viability was assayed 5 days following treatment using CellTiter-Glo. Statistical analyses were performed comparing filtered supernatants from transfected cell to filtered supernatants from control 293T cell treatments, and likewise for the unfiltered groups. As shown in Figure 2A, only CEM cells treated with supernatants from VP40-transfected 293T cells showed significant reduction in viability. However, supernatants from NP- or GP-transfected cells had minimal effect on CEM cell viability. Jurkat T-cells showed significant decreases in viability following treatments with VP40 and NP transfection supernatants in contrast to treatment with GP-transfected cell supernatants (Figure 2B). Interestingly, CEM cells are derived from the T lymphoblast whereas Jurkat cells originate from the more differentiated peripheral blood T lymphocyte. As such, the differential dysregulation seen between these two cell types may be an indication of selective susceptibility of T cells to Ebola structural proteins based on their stage of development. Finally, U937 monocytic cells showed significant reduction in viability when treated with any of the three supernatants, with VP40-transfected supernatants showing the most pronounced effect (Figure 2C). These results suggest that monocytes, in contrast to T cells, are particularly susceptible to Ebola protein-transfected cell supernatants. When CEM, Jurkat, and U937 cells were treated with supernatants from 293T cells that were transfected with multiple plasmid combinations (i.e., VP40 + GP, VP40 + NP, GP + NP, and VP40 + GP + NP), all three immune cell types showed significant decreases in viability from the triple combination of VP40 + GP + NP while only the Jurkat cells showed significant decreases from treatment with supernatants from all transfection doublets (data not shown). Furthermore, antibiotic alone (Hygromycin B, Geneticin, and Zeocin) treated supernatants of 293T cells (5 days) did not alter cell viability of immune cells (data not shown).

As VP40 alone or VP40 + GP ± NP can form VLPs, we next asked whether the specific size of the material in supernatants of transfected cells contributed to the observed decrease in viability. VLPs are distinct from other extracellular vesicles, which can be differentiated based on a number of characteristics, including size and surface markers. VLPs are 400 nm in size or larger, similar to EBOV virions (Bavari et al., 2002; Noda et al., 2002; Warfield et al., 2003), whereas extracellular vesicles (i.e., exosomes) are smaller than 220 nm, and typically approximately 100 nm (Fleming et al., 2014; Sampey et al., 2014). For this reason, we passed transfected cell supernatants through a 0.22 micron filter and asked whether the same phenotype in treated immune cells could be observed compared to those treated with unfiltered supernatants. A side-by-side comparison of filtered versus unfiltered supernatants showed similar effects on all cell types (Figures 2A–C). Since filtering did not abrogate the earlier observed effects, we concluded that it was unlikely that the detrimental agent within the transfected cell supernatant was Ebola VLPs or cellular apoptotic bodies that are also larger than 400 nm (Akers et al., 2013; Schwab et al., 2015). Additionally, these experiments indicated that the factor(s) effecting the apoptosis was not free VP40 protein, as purified VP40 showed little effect on U937 cell viability (Figure 1B). Collectively, these results imply that supernatants from Ebola protein-transfected 293T cells may be capable of negatively impacting both T cells and monocytes.

As the supernatants from cells transfected with VP40 DNA appeared to pose greater toxicity to monocytes than to T lymphocytes, we wished to explore further the effect of VP40 on differentiated macrophages. This was of interest since EBOV naturally targets monocytes and especially macrophages in vitro and in vivo during early infection (Jaax et al., 1996; Ryabchikova et al., 1999; Geisbert et al., 2003; Falasca et al., 2015; Messaoudi and Basler, 2015; Messaoudi et al., 2015; Nyakatura et al., 2015; Singh et al., 2015). We differentiated U937 monocytes into macrophages using phorbol 12-myristate 13-acetate (PMA) for 5 days, followed by treatment with purified VP40 protein, as well as unfiltered and filtered VP40 transfection supernatants. Differentiated cells were incubated for 5 days and cell viability was measured using CellTiter-Glo. While purified VP40 protein alone had no effect on differentiated U937 cell viability, both filtered and unfiltered cell-free media from VP40-transfected cells resulted in significantly decreased cellular viability (Figure 2D).

Next, we differentiated primary monocytes into macrophages using PMA for 2 days and treated them with filtered supernatant from control 293T cells, unfiltered or filtered supernatants from VP40-transfected 293T cells, or filtered supernatants from Ebola VP40-Transfected Resistant 293T Clone (EVTR2C) cell supernatants. EVTR2C cells are a clone of VP40-transfected 293T cells resistant to the effects of VP40. These resistant clones are a stable population of 100% VP40-transfected 293T cells under Hygromycin B selection. We believe that EVTR2C cells are able to survive stably with the production of a lower level of VP40 protein in comparison to normal VP40-transfected 293T cells (see Materials and Methods). Differentiated primary cells were incubated for 2 days and cell viability was measured using CellTiter-Glo. The primary cells were only treated for 2 days with cell supernatants as opposed to the 5-day treatment received by the U937 cells. Viability of the treated primary cells was noticeably decreasing over time upon visual inspection. Strikingly, PMA-activated primary monocyte viability was drastically reduced with the treatment of unfiltered and filtered VP40-transfected 293T and filtered EVTR2C cell supernatants in comparison to the control treatment (Figure 2E). This further confirms that free, purified VP40 protein alone exhibits no toxicity to monocytes; however, primary monocytes that have differentiated into macrophages are more susceptible to supernatant from VP40-producing cells, compared to macrophages derived from monocyte cell lines. Collectively, these data suggest that VP40 produced from transfected cells can potentially be packaged into protected vesicles, which in turn can exert detrimental effects on immune recipient cells.

We next asked whether VP40 produced in the transfected cells was incorporated into exosomes, potentially affecting recipient cells. The packaging of viral proteins into exosomes has been previously shown in many other infections including HIV-1, HTLV-1 and Rift Valley fever viruses (Meckes et al., 2013; Fleming et al., 2014; Jaworski et al., 2014a; Sampey et al., 2014; Ahsan et al., 2016). Incorporation into exosomes protects the packaged proteins and nucleic acids from degradation by extracellular enzymes and allows them to reach recipient cells. Therefore, we concentrated vesicles from the supernatants of transfected cells using CD63 Dynabeads and NT80/82 nanotrap particles (NTs), which have previously been shown to effectively capture exosomes from dilute supernatants (Jaworski et al., 2014a,b; Sampey et al., 2016). CD63 is a tetraspanin marker abundantly expressed on exosomes and also within virally infected cells (Sampey et al., 2016). Additionally, we used Dynabeads alone as a negative control, and NT229 beads which we had observed to be a strong binder of free VP40 protein (see data in Figure 3C). Filtered supernatants from VP40-transfected 293T cells were treated with nanoparticle beads, washed, resuspended in SDS Laemmli buffer, and analyzed by Western blot for the presence of VP40 protein. VP40 was present in the samples immunoprecipitated with CD63 and NT80/82 beads (Figure 3A, lanes 2 and 4), but was not precipitated with Dynabeads alone, or with NT229 beads that capture purified VP40 (Figure 3A, lanes 3 and 5). It is interesting to note that the VP40 from exosomes exhibited a slightly higher molecular weight, indicating selective incorporation of post-translationally modified VP40 (i.e., phosphorylation; p-VP40) into exosomes.

We have previously shown that specific nanoparticles can capture viral proteins, i.e., HIV-1 Tat (Narayanan et al., 2013; Jaworski et al., 2014b). Thus, we asked whether free VP40 could be captured with any of the newer set of 9 particles or nanotraps described in Figure 3B. We hypothesized that there could be free VP40, exosome-associated VP40, VLP-associated VP40, or virally associated VP40 that could be captured using these nanoparticles, presenting a potential diagnostic tool of EBOV disease progression. We investigated this by treating purified VP40 protein with nine different nanotraps (NTs 210, 212, 213, 217, 219, 222, 223, 224, and 229), some of which were previously shown to effectively capture HIV-1 viral proteins (Narayanan et al., 2013; Jaworski et al., 2014a,b), under two different binding conditions: a phosphatases-buffered saline (PBS) buffer that contained no SDS, and a second buffer that contained SDS (1:4; Radioimmunoprecipitation assay (RIPA) and Laemmli buffer). When using PBS alone, almost all particles, except NT213, showed varying levels of bound VP40, with the NT229 being the strongest binder of free VP40 (Figure 3C). However, when we used SDS buffer, we allowed the nanotraps to bind to VLPs in a mixture of RIPA and Laemmli buffer overnight at 4°C. The next day, samples were washed, isolated on a 4–20% SDS/PAGE, and subsequently analyzed by Western blot for VP40 and GP proteins. The bottom panels in Figure 3C show that in the presence of SDS buffer, NT219 was the strongest binder of Ebola proteins from VLP, whereas NT229 showed minimal or no binding in this assay.

We finally asked whether VLPs spiked into bodily fluid could be captured with NT219 beads under these strong denaturing conditions. To that end, we spiked human saliva, urine, and serum with VLPs and treated the samples with SDS buffer (1:1; Laemmli to RIPA). A 30% slurry of NT219 beads was added, samples were incubated overnight at 4°C, washed with TNE50 + 0.1% NP40 the next day and analyzed by Western blot for presence of VP40 protein. As shown in Figure 3D, NT219 beads were able to bind to VP40 in both saliva and urine samples under these reducing conditions. The VP40 from saliva samples contained various degraded smaller forms, whereas the urine sample showed a single band for the protein (Figure 3D, lanes 4 and 6). The binding efficiency was approximately 1% under these conditions as determined by densitometry (data not shown). Interestingly we observed no VP40 present from the serum samples spiked with the VLP, indicating that the proteins may have been rapidly degraded even when using SDS buffer. Collectively, these data indicate that exosome-associated VP40 can be captured with either CD63 beads or nanoparticles from transfected supernatants, and that the use of nanotraps in an SDS buffer (to disrupt VLPs, and by analogy virions) may be an alternative method in concentrating inactive Ebola proteins or potentially nucleic acids for downstream diagnostic assays.

Our previous results in Figure 3A indicated that the VP40 incorporated into exosomes was of a slightly higher molecular weight, suggesting a selective post-translational modification. It has previously been shown that VP40 is phosphorylated at tyrosine 13 (Y¹³) by c-Abl1 kinase, which was necessary for the productive replication of high titer EBOV in vitro (García et al., 2012). Therefore, we asked whether VP40 could potentially be phosphorylated in cells prior to packaging and release with exosomes. The VP40 amino acid sequence contains multiple potential sites of phosphorylation. Using our mass spectrometry data of EBOV virions we found Ser-233, Thr-272, Thr-277, and Ser-278 to be potentially phosphorylated (Figures 4A,B). The whole phosphoproteomic analysis of EBOV is ongoing and will be published elsewhere (data not shown). The whole phosphoproteomic analysis of EBOV is ongoing and will be published elsewhere (data not shown). Interestingly, Ser-233 of VP40 is part of a ²³³SPEKI²³⁷ sequence (shown in red; Figure 4A) which resembles the Cyclin-dependent kinase 2 (Cdk2) phosphorylation motif (S/T)PX(K/R) (Deng et al., 2002). To determine if Cdk2 is indeed involved in the phosphorylation of VP40, we designed in vitro kinase assays using cell extracts from which Cdk2 was immunoprecipitated with α-Cdk2 antibody. In addition, antibodies against Cyclin A and Cyclin E (α-CycA and α-CycE), which are normal partners of Cdk2, were utilized to precipitate specific Cdk2 kinase complexes. In vitro kinase assays were then performed using purified VP40 protein and [γ-³²P] ATP. Results in Figure 4C indicate that there is a dramatic increase in VP40 phosphorylation in the presence of Cdk2 as compared to controls (lanes 1-3). VP40 phosphorylation additionally increased when the kinase was precipitated with either Cyclin E- or Cyclin A-specific antibodies (Figure 4C, lanes 4 and 5). This is consistent with the association of the Cyclins with Cdk2 in an active kinase complex, rather than Cdk2 alone which may represent a mixture of inactive complex (Cdk2 alone) with active complexes (Cdk2/CycE, Cdk2/CycA) in cells.

As further confirmation of the involvement of Cdk2, we then asked whether the phosphorylation of VP40 in vitro could be prevented with specific Cdk inhibitors. Cdk2 inhibitors with high selectivity such as Alsterpaullone, Indirubin-3′-monoxime, Purvalanol A, and r-Roscovitine have previously been shown to inhibit the replication of other viruses including HIV-1, Hepatitis C Virus, Human Cytomegalovirus, Adenovirus, and Varicella-Zoster Virus (Bresnahan et al., 1997; Taylor et al., 2004; Guendel et al., 2010; Cheng et al., 2013; Munakata et al., 2014; Roy et al., 2015). We therefore investigated if any of these inhibitors could affect the phosphorylation of VP40 in vitro. While Alsterpaullone and Indirubin-3′-monoxime resulted in only a moderate down-regulation of VP40 phosphorylation (Figure 4C, lanes 6 and 7), Purvalanol A was slightly more effective (Figure 4C, lane 8), and importantly, r-Roscovitine completely inhibited the phosphorylation of VP40 (Figure 4C, lane 9). Collectively, these data indicate that Cdk2/Cyclin E and Cyclin A complexes (markers of late G1 and early S phases of cell cycle) efficiently phosphorylate VP40 in vitro, and this phosphorylation can be prevented with specific Cdk inhibitors.

Cdk2/Cyclin E complexes are most active during the G1/S phase of the cell cycle, however, most cell lines used in vitro (i.e., 293T cells) are a mixed population of cells at the G1 (∼65%), S (∼20%), and G2/M (∼10%) phases. To obtain a more uniform population of cells at G1/S, we treated cells with Hydroxyurea (Hu), which is a reversible compound that targets early S phase DNA synthesis (Kashanchi et al., 2000). We transfected 293T cells with VP40 plasmid and added Hygromycin B for plasmid selection. Cells were either untreated or blocked at the G1/S phase with Hydroxyurea (2 mM) for 1 day. Cells were subsequently pulsed with radio-labeled [γ-³²P] ATP and treated with r-Roscovitine for 4 h in complete media, followed by washes and immunoprecipitations using α-VP40 antibody. The data in Figure 4D show that VP40 is indeed phosphorylated in vivo and its phosphorylation is inhibited by r-Roscovitine treatment. Kinase activity as a percentage is quantified using densitometry as illustrated in the panels beneath Figures 4C,D.

In order to confirm the specific site of phosphorylation on VP40, we utilized five 10–12 mer specific peptides that span various residues that are potentially modified (as shown in Figures 4A,B). The expected target of Cdk2 phosphorylation was Ser 233, which is part of a SPEKI sequence that resembles Cdk2’s (S/T)PX(K/R) consensus phosphorylation site (Deng et al., 2002). The ADLTSPEKIQAI peptide (S3) and the S233A mutated ADLTAPEKIQAI peptide (S3M) were used in in vitro kinase assays. The Cyclin E-precipitated Cdk2 kinase complex was incubated with the peptide substrates and phosphorylation was detected using Whatman glass microfibre filters. Other potential targets were also examined using additional synthesized peptides containing Thr-277 (GKKVTAKNGQPI, T7), Ser-278 (GKKVASKNGQPI, S8), and Thr-272 (LVHKLTGKKV, T2) residues (Figure 4E). To distinguish between Thr-277 and Ser-278 residues, alanine mutations were introduced: S278A for T7 and T277A for S8. The S3 peptide containing the putative Cdk2 phosphorylation site showed the greatest level of phosphorylation by Cdk2/Cyclin E, which was abrogated in the S3M mutant (Figure 4E). Peptide T7 containing the Thr-277 site showed considerably reduced phosphorylation, and the peptides containing Thr-272 (T2) and Ser-278 (S8) showed only background levels of phosphorylation, similar to S3M peptide (Figure 4E). IP experiments were conducted using CEM extracts as we have previously shown that these cell extracts contain active Cdk/Cyclin kinases that can easily be purified with specific antibodies (Wang et al., 2001). Taken together, these results suggest that VP40’s Ser-233 is the target site for phosphorylation by Cdk2/Cyclin E.

It has previously been shown that a downregulation of various microRNA machinery components such as Dicer, Drosha, Exportin 5 and Ago proteins have been linked to the induction of apoptosis (Su et al., 2009; Han et al., 2013; Bian et al., 2014; Lombard et al., 2015), while upregulation of Ago 1 has been linked to a slowing down of the cell cycle and eventual apoptosis (Su et al., 2009; Parisi et al., 2011: 1). Given that we observed apoptosis in cells treated with purified VP40 protein and supernatants from cells transfected with VP40 DNA, we next asked whether Ebola VP40 was able to affect the cellular miRNA machinery of transfected cells, as well as cells receiving exosome-laden transfected cell supernatant treatments. It has been previously shown that some viruses interfere with host miRNA machinery as a means of evading host immune responses important for cell signaling and regulation of apoptosis (Coley et al., 2010; Van Duyne et al., 2012). Additionally, it has been observed that EBOV proteins VP35, VP30, and potentially VP40 are capable of deregulating the host RNAi pathway as suppressors of RNA silencing (Fabozzi et al., 2011). Here, 293T cells were transfected with plasmids producing VP40 and kept under Hygromycin B antibiotic selection. Cell pellets were harvested, lysed, and analyzed by Western blot for levels of Dicer, Drosha, Ago 1, Exportin 5, DGCR8, TRBP, VP40 and Actin. Results in Figure 5A show a down-regulation of Dicer, Drosha, Ago 1, and DGCR8 protein levels in cells transfected with VP40 DNA in comparison to mock transfected cells. However, the levels of Exportin 5 remained constant while TRBP levels slightly increased.

We next examined the effects of exosomes from transfected cells on recipient cell miRNA machinery. Exosomes were purified from VP40-transfected cells using a 0.22 micron filter and ultracentrifugation, followed by treatment of recipient CEM cells with varying concentrations of exosomes (0.3, 0.75, and 1.5 mU AChE). Recipient cells were treated for 5 days, harvested, and analyzed by Western blot for the components of the miRNA machinery, along with accessory proteins including TRBP and DGCR8. Interestingly, a similar decrease was observed in the expression of Dicer, Drosha and Ago 1 in lymphocytes treated with the highest concentration of exosomes (Figure 5B). TRBP, DGCR8, and Exportin 5, on the other hand, were unaffected by VP40-associated exosomes. Together, these data suggest that VP40 may be capable of dysregulating some specific components of miRNA machinery components not only in infected donor cells, but also in naïve neighboring cells receiving VP40-containing exosomes.

We next asked whether treatment of VP40-transfected 293T cells with FDA-approved drugs would be able to down-regulate the release of exosomes, and thereby potentially mitigate the detrimental effects seen in recipient cells. We screened a panel of FDA- and non-FDA-approved drugs to find that Esomeprazole, Oxytetracycline, and Cambinol reduce exosomal release in HIV-1 infected cells (unpublished data). Here, 293T cells were transfected with VP40 DNA, placed under antibiotic selection, and treated with increasing concentrations of 1 nM to 10 μM of Esomeprazole, Oxytetracyline, Cambinol, or DMSO as control. Cells were then pelleted, lysed, and analyzed by Western blot for classic exosomal markers CD63 and Alix, as well as Actin. Results in Figure 6A show an increase in CD63 and Actin in cells transfected with VP40 DNA in comparison to untransfected controls (lanes 1 and 2). In addition, a decrease in CD63 and Actin was observed in transfected cells treated with increasing concentrations of Oxytetracycline (Figure 6A, lanes 6–8). Treatment of cells with increasing concentrations of Esomeprazol, Cambinol, and DMSO resulted in no significant differences in CD63, Alix, or Actin levels.

As Oxytetracycline showed a positive effect of potentially down-regulating exosomal release from donor cells, we then asked whether this same treatment on donor cells would reduce the inhibitory effects we observed on recipient immune cells. We placed our VP40-resistant EVTR2C cells under varying concentrations (100 nm-10 μM) of Oxytetracycline and incubated for 5 days. Supernatants were harvested, filtered through a 0.22 micron filter, and then used to treat CEM, Jurkat, or U937 recipient cells. Treatment of immune cells with untreated, filtered 293T cell supernatant served as a negative control. Cell viability was then assayed with CellTiter-Glo. Results in Figures 6B,D show an expected decrease in cell viability in the three cell types treated with EVTR2C supernatant. Interestingly, CEM and Jurkat cells treated with 0.1, 1.0, and 10 μM Oxytetracycline showed a corresponding increase in cell viability in a dose-dependent manner (Figures 6B,C). U937 cells showed no significant change in cell viability until treatment with the highest dose of Oxytetracycline (Figure 6D). Collectively, these data indicate that an increase in exosomal release from cells may occur in the presence of VP40. Additionally, treatment with Oxytetracycline may cause a reduction of exosomal release in VP40-producing cells, and thereby diminish the inhibitory effects on neighboring recipient immune cells.

We have previously shown that HIV-1-infected cells have increased levels of cytosolic unprocessed CD63, while exosomes from HIV-1-infected cells contain increased levels of glycosylated CD63, indicating that infection results in increased CD63 production or exosome biogenesis (Narayanan et al., 2013; Sampey et al., 2016). We likewise observed a similar increase in the levels of CD63 in exosomes in presence of VP40 (Figure 6A, lane 2). To further characterize this change, we used NT80/82 beads to concentrate the exosomes in supernatants from 293T and EVTR2C cells that were simultaneously harvested and lysed. Both exosomes and cellular extracts were analyzed by Western blot for levels of CD63, Alix, and Actin. Results in Figure 7A (left panel) show an increase in CD63 levels in the presence of VP40 compared to control cells. However, the intracellular levels of glycosylated CD63 and Alix remained similar. In contrast, Alix and glycosylated CD63 increased in exosomes from EVTR2C cells in comparison to controls (Figure 7A, right panel). These data indicate that VP40 may induce an increased total level of CD63 or an increased production of exosomes, similar to the HIV-1-infected cell phenotype.

As exosomal biogenesis and release is governed largely by the ESCRT pathway, we next asked if components of the ESCRT machinery were being affected by the presence of VP40 in our resistant clones. EVTR2C and 293T cells were cultured to mid-log phase, harvested, and lysed. Whole cell extracts were analyzed by Western blot for levels of ESCRT I (TSG101), II (EAP20, EAP45), and III (CHMP6) proteins, as well as VP40. The data in Figure 7B show an increase in levels of TSG101, EAP20, and EAP45 proteins in EVTR2C cells in comparison to 293T cells. Western blot also confirmed the presence of VP40 in EVTR2C cells. Finally, as we had observed a decrease in exosomal release with Oxytetracycline treatment (Figure 6A, lanes 6–8), we hypothesized that ESCRT components may be dysregulated by this drug treatment. We treated EVTR2C cells with Oxytetracycline for 5 days followed by lysis and Western blot for ESCRT I–III protein components, as well as VP40 protein and Actin. Data in Figure 7B (lane 3) shows that treatment with Oxytetracycline resulted in a downregulation of ESCRT III protein CHMP6. Surprisingly, levels of intracellular VP40 were also decreased in EVTR2C cells treated with Oxytetracycline. Together, these results indicate that exosomal markers such as CD63 and specific ESCRT machinery components may be upregulated in VP40-producing cells, correlating with a possible increase in exosome production. Treatment of VP40-containing cells with Oxytetracycline may also offer a potential tool for decreasing the output of exosomes through inhibition of a CHMP6-mediated pathway, or by inhibiting the production or accumulation of VP40 within cells.