Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

Abstract

Background: Ebola virus (EBOV) mainly targets myeloid cells; however, extensive death of T cells is often observed in lethal infections. We have previously shown that EBOV VP40 in exosomes causes recipient immune cell death.

Methods: Using VP40-producing clones, we analyzed donor cell cycle, extracellular vesicle (EV) biogenesis, and recipient immune cell death. Transcription of cyclin D1 and nuclear localization of VP40 were examined via kinase and chromatin immunoprecipitation assays. Extracellular vesicle contents were characterized by mass spectrometry, cytokine array, and western blot. Biosafety level-4 facilities were used for wild-type Ebola virus infection studies.

Results: VP40 EVs induced apoptosis in recipient T cells and monocytes. VP40 clones were accelerated in growth due to cyclin D1 upregulation, and nuclear VP40 was found bound to the cyclin D1 promoter. Accelerated cell cycling was related to EV biogenesis, resulting in fewer but larger EVs. VP40 EV contents were enriched in ribonucleic acid-binding proteins and cytokines (interleukin-15, transforming growth factor-β1, and interferon-γ). Finally, EBOV-infected cell and animal EVs contained VP40, nucleoprotein, and glycoprotein.

Conclusions: Nuclear VP40 upregulates cyclin D1 levels, resulting in dysregulated cell cycle and EV biogenesis. Packaging of cytokines and EBOV proteins into EVs from infected cells may be responsible for the decimation of immune cells during EBOV pathogenesis.

Figure 1.

Cell cycle analysis of VP40 clones. 293T, V2CL, and V2CH cells were seeded in a 96-well plate at 5 × 10⁵ cells in 100 μL of fresh media. Cells were blocked at G₀ (starvation; 0.1% fetal bovine serum DMEM), G₁/S (20 mM of hydroxyurea), and G₂/M (18-hour 20 mM of hydroxyurea pretreatment, followed by release for 1 hour, and subsequent treatment with 50 ng/mL of nocodazole) for 2 days. Blocked cells were then imaged (A) and assayed for cell viability with CellTiter-Glo (B). The same experiment was repeated; however, cells were allowed to incubate for 5 days after blocking. Cells were subsequently imaged (C) and assayed for cell viability via CellTiter-Glo (D). Statistical analysis by Student’s 2-tailed t test compares V2CL and V2CH cell cycle-blocked groups with corresponding 293T groups (†, P < .05; ††, P < .01; †††, P < .001). Additional Student’s 2-tailed t test compares cell cycle-blocked groups with controls of their own cell type (**, P < .01; ***, P < .001).

Figure 2.

Effect of nuclear VP40 on cyclin and cdk regulation and activity. (A) Log phase 293T, V2CL, V2CI, and V2CH cells were harvested, lysed, and subjected to SDS/PAGE for western blot analysis of cyclin D1 (CycD1), cyclin E (CycE), cyclin A (CycA), cyclin B1 (CycB1), cdk4, cdk6, cdk2, cdk1, and actin levels. (B) Five hundred micrograms of 293T or V2CH whole-cell extracts were used for IP with 10 µg of either normal rabbit immunoglobulin G (IgG) or α-CycD1. IPed material was incubated with 50 µL 30% of Protein A/G for 2 hours, followed by two 1× PBS washes and 1 kinase buffer wash. Pellets were resuspended in kinase buffer, and 15 µL samples were incubated with 50 µg of either no peptide, 780S, or 780A peptide, along with 2 µL of [γ-³²P] ATP. Some samples were also treated with Fascaplysin ([Fascap.] 1 µM). All samples incubated for 1 hour, followed by dotting onto Whatman glass microfiber filters and drying for 30 minutes. Filters were incubated in 1× TE buffer with gentle agitation for 2 days, dried, and then quantified with a scintillation counter. Background levels of CycD1 kinase activity (α-CycD1 IP with 780A peptide substrate) are indicated by black bar. (C) Log phase 293T and V2CH cells were harvested for separation of cytoplasmic (Cyt) and nuclear (Nuc) compartments with the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Fisher Scientific). Western blot analysis for levels of VP40, CHMP6, HDAC1, and Actin was performed. (D) Log phase 293T and V2CH cells (3 × 10⁶) were harvested and cross-linked with 1% formaldehyde for 1 hour followed by quenching with 1.25 glycine (9:1 cell suspension/glycine). Samples were sonicated, and 100 µL of each sample (corresponding to approximately 5 × 10⁵ cells) was used for IP with 1 µg of anti-Pol II, α-p300, or α-VP40 at 4°C overnight. The next day, Protein A/G (30% slurry) was added and incubated for 2 hours at 4°C. Complexes were washed once with each TNE300 + 0.1% NP40, TNE150 + 0.1% NP40, TNE50 + 0.1% NP40, and IP wash buffer before the addition of proteinase K (800 units/mL). Samples were incubated for 15 minutes at 65°C, reversing solution was added, and samples were incubated for an additional 90 minutes at 65°C. DNA was purified, and qPCR was performed with 2 μL of undiluted DNA with primers for the CycD1 promoter region (spanning −180 to +238 from the messenger RNA start site at +1). The absolute quantification of the samples was determined based on the cycle threshold value relative to the standard curve generated from serial dilutions of DNA from 293T cells. Data are presented as percentage (%) of the input (DNA purified from sonicated samples before IP with specific antibodies). Student’s 2-tailed t test compares V2CH ChIPed DNA with corresponding 293T ChIP samples (**, P < .01).

Figure 3.

Alteration of cell viability by VP40 in multiple cell types. Cells including (A) U937, HeLa, and (B) 3 peripheral blood mononuclear cells (PBMCs) log-phase cultures (~1.65 × 10⁵ cells) were transfected with attractene and 1.5 µg of cytomegalovirus-VP40 plasmid. The PBMCs received a 1-time treatment of 50 IU/mL of interleukin-2 the day before transfection. Control cells received attractene treatment alone. Cell viability was assayed 3 days post-transfection. Statistical analysis by Student’s 2-tailed t test compares control cells with transfected cells (*, P < .05; **, P < .01).

Figure 4.

Differential biogenesis of exosomes at different phases of the cell cycle. 293T and V2CL cells were blocked at G₀ (starvation; 0.1% fetal bovine serum DMEM), G₁/S (20 mM of hydroxyurea), and G₂/M (18-hour 20 mM of hydroxyurea pretreatment, followed by release for 1 hour and subsequent treatment with 50 ng/mL of nocodazole) for 5 days. Control (unsynchronized) cells were also incubated for 5 days. Black arrows point to bands of noticeable difference between V2CL and 293T cells. (A) Blocked cells and controls were harvested, washed twice in 1× PBS, and lysed. Samples were run on a 4–20% Tris-glycine gel and analyzed by western blot for the presence of ESCRT pathway proteins (VPS4, EAP45, TSG101, CHMP6, and EAP20), exosomal markers (Alix and CD63), and Actin. (B) Cell-free supernatants from blocked cells were harvested and passed through a 0.22-µm filter. One milliliter of filtered supernatant was incubated with 30 μL of NT80/82 particles overnight at 4°C. The next day, the NT pellet was washed once in 1× PBS and resuspended in 10 μL Laemmli buffer, followed by SDS/PAGE and western blot analysis for VP40 protein, exosomal markers CD63 and Alix, and Actin. (C) One milliliter of filtered supernatant from blocked and control cells was incubated with 30 μL of NT80/82 particles overnight at 4°C. The next day, the NT pellets were isolated, washed, and subjected to AChE assay for quantification of exosomes. Statistical analysis was completed by Student’s 2-tailed t test (*, P < .05; ***, P < .001).

Figure 5.

Extracellular vesicles released by VP40-producing cells at different phases of the cell cycle. 293T, V2CL, and V2CH cells were blocked at G₀ (starvation; 0.1% fetal bovine serum DMEM), G₁/S (20 mM of hydroxyurea), and G₂/M (18-hour 20 mM of hydroxyurea pretreatment, followed by release for 1 hour and subsequent treatment with 50 ng/mL of nocodazole) for 5 days. Supernatants were harvested, filtered (0.22 μm), and analyzed by ZetaView for size (peak [mode] diameter) (A) and concentration of particles (B). Statistical analysis by Student’s 2-tailed t test compares V2CL and V2CH cell cycle-blocked groups with corresponding 293T groups (†, P < .05; ††, P < .01; †††, P < .001). Additional Student’s 2-tailed t test compares cell cycle-blocked groups with controls of their own cell type (*, P < .05; **, P < .01; ***, P < .001).

Figure 6.

Iodixanol gradient separation of extracellular vesicles (EVs) from 293T and VP40-producing cells. 293T and V2CI cells were grown in exosome-free media for 5 days, followed by harvesting of the supernatant and incubation with ExoMAX (1:1 reagent/filtered supernatant) reagent overnight at 4°C. The EVs were pelleted, resuspended in 300 µL of sterile 1× PBS, and loaded onto a 6–18% iodixanol density gradient (1.2% increments). Samples were ultracentrifuged for 90 minutes at 100000 ×g, followed by harvesting and isolation of each fraction, and incubation with 30 µL of NT80/82 particles overnight at 4°C. The NT pellets were washed in 1× PBS, resuspended in 12 µL of Laemmli buffer, and loaded onto a 4–20% Tris-glycine gel. Western blot of 293T (A) and V2CI (B) fractions were analyzed for levels of VP40, CD63, CD81, CD9, and Actin. Major groups of EVs or exosome type are indicated by black boxes.

Figure 7.

The presence of VP40 in exosomes in in vitro and in vivo EBOV-infected cells. (A) HUVECs were cultured and infected with EBOV (MOI of 1) and incubated for 3 days under BSL-4 containment. Two milliliters supernatant were harvested, passed through a 0.22-µm filter, and incubated with ExoMAX (1:1 reagent/filtered supernatant) reagent overnight at 4°C. EVs were pelleted, resuspended in 0.5 mL 1× PBS, and loaded on qEV columns. Fraction numbers 7–10 (0.5 mL each) were collected and separately incubated with 30 μL NT80/82 at room temperature for 1 hour. The EV-bound NTs were washed with 1× PBS, followed by resuspension in 10 μL 2× NuPAGE LDS sample buffer, heating at 95°C for 10 minutes, and loading onto a 4–12% Tris-glycine gel for subsequent western blot analysis for VP40, GP, NP, and Actin levels. Negative control (Null) samples consisted of purified exosomes from uninfected HUVECs. (B) Gamma-irradiated and inactivated NHP (rhesus monkey) serum samples were obtained. NHP 1: day 0 prebleed sample. NHP 2: pool of day 4 and day 5 postinfection (pi); NHP 2 died on day 7 post-EBOV infection. NHP 3: pool of day 8–11 pi; NHP 3 died on day 12 post-EBOV infection. One hundred microliters of serum were diluted with 400 μL sterile 1× PBS and filtered (0.22 µm). Twenty-five microliters NT80/82 particles were incubated with the filtered samples at 4°C overnight. The next day, NT pellets were washed once in 1× PBS, resuspended in 12 μL Laemmli buffer, run on 4–20% SDS/PAGE, and analyzed by western blot for VP40 protein and exosomal markers CD81 and CD9.

Figure 8.

Recipient monocyte and T-cell apoptosis. (A) Supernatants from 293T (Control), V2CL, and V2CH cells (5 days, 0.22 µm filtered, in exosome-free media) were used to treat CEM, Jurkat, U937, and 293T recipient cells. Recipient cells were seeded in a 96-well plate at 5 × 10⁵ cells in 50 μL of fresh media, treated with 50 μL of filtered supernatants, and incubated 5 days, followed by analysis by CellTiter-Glo for cell viability. Statistical analysis by Student’s 2-tailed t test compares supernatant-treated groups with control cells of the same type (*, P < .05; **, P < .01; ***, P < .001). 293T, V2CL, CEM, Jurkat, and U937 cells were grown in exosome-free media for 5 days, followed by harvesting and filtering (0.22 µm) of the supernatant. Log-phase CEM, Jurkat, and U937 cells were plated at a density of 1 × 10⁶ cells/mL and treated with increasing concentrations (100, 250, and 500 μL) of supernatant from either 293T, V2CL, or their own cell type. Cells were incubated for 5 days, followed by harvesting of the cells and lysis. Lysates of CEM (B), Jurkat (C), and U937 (D) cells were then run on a 4–20% Tris-glycine gel and subjected to western blot analysis for apoptotic markers procaspase 3 and PARP-1 and their cleaved forms, and Actin.

Figure 9.

Induction of recipient T-cell death by purified VP40 EVs. (A) 293T and V2CH cells were grown in exosome-free media for 5 days, followed by harvesting of cell-free supernatants and filtration through 0.22 μm. Supernatants were then spun at 100000 ×g for 90 minutes to pellet EVs, followed by resuspension in sterile 1× PBS. Concentrations of resulting ultracentrifuged EVs were determined with ZetaView analysis, followed by treatment of CEM cells with increasing concentrations of EVs (10000, 25000, or 75000 particles/cell) from V2CH cell type. Controls included CEM cells that were left untreated and CEM cells that received a treatment of the highest concentration of 293T cells (75000 particles/cell). Cells were incubated for 3 days followed by analysis of cell viability by CellTiter-Glo. (B) 293T and V2CI cells were grown in exosome-free media for 5 days. Cell-free supernatants were harvested and incubated with equal volumes of ExoMAX overnight at 4°C. The EVs were pelleted, resuspended in 400 µL sterile 1× PBS, and loaded onto a 6–18% iodixanol density gradient (1.2% increments). Samples were ultracentrifuged for 90 minutes at 100000 ×g, followed by harvesting and isolation of each fraction. Select fractions (13.2 + 14.4 and 16.8 + 18.0) were pooled and subjected to a second ultracentrifuge spin for 90 minutes at 100000 ×g diluted in 1× PBS to pellet the EVs away from residual iodixanol. Resulting EV pellets were resuspended in 100 µL sterile 1× PBS and used to treat recipient CEM cells at a concentration of 10000 particles per cell (concentrations determined by ZetaView analysis). Cells were incubated for 5 days followed by analysis of viability by CellTiter-Glo assay. Statistical analysis by Student’s 2-tailed t test compares groups treated with EVs from V2C cells to those treated with EVs from 293T cells (*, P < .05; ***, P < .001).

Figure 10.

Effect of cdk4/6 inhibitors on VP40-producing cell viability and extracellular vesicle biogenesis. 293T and V2CH cells were treated with low, medium, and high concentrations of Fascaplysin (0.1, 0.5, 1.0 µM) or Ribociclib (0.1, 1.0, 10.0 µM) for 5 days. DMSO was also used at a final concentration of 1%. (A) Supernatants were harvested, filtered (0.22 µm), and analyzed by ZetaView for concentration of particles. (B) Cell viability was also measured by CellTiter-Glo. Statistical analysis by Student’s 2-tailed t test compares drug-treated groups to untreated controls of their own cell type (*, P < .05; **, P < .01; ***, P < .001).