KEYWORDS: chemokines, cytokines, Ebola virus, filovirus, host response, interferons, macrophages, Reston virus, Toll-like receptor 4, Toll-like receptors

RESTV infection is not associated with strong changes in gene expression.

To compare the host response to infection with highly pathogenic and presumably nonpathogenic ebolaviruses, human MDMs were infected with purified, endotoxin-free EBOV or RESTV particles at a multiplicity of infection (MOI) of 5 to 7. As controls, cells were treated with lipopolysaccharide (LPS) or were mock infected. Samples were prepared at different time points starting at 6 h up to 4 days postinfection (dpi). At the desired time points, total cellular RNA was isolated for transcriptional analysis (RNA sequencing [RNA-Seq] and quantitative reverse transcription-PCR [qRT-PCR]) and cell supernatant cytokine concentrations were determined by Luminex analysis. Immunofluorescence analysis was performed to visualize infected cells.

We detected viral inclusions indicative of established infection by immunofluorescence analysis in both EBOV- and RESTV-infected MDMs at comparable rates (about 65%) at 1 dpi (Fig. 1A andB). About 90% of the cells were infected with EBOV or RESTV at 4 dpi (data not shown). For a quantitative comparison of infection, we analyzed VP35 mRNA levels. Alignment of viral RNA reads generated by RNA-Seq and qRT-PCR analysis was performed on mRNA isolated from EBOV- and RESTV-infected MDMs. While the amount of VP35 mRNA was lower in RESTV-infected MDMs than in EBOV-infected cells at 6 h postinfection (hpi), the observed difference was not statistically significant (Fig. 1C andD). Consistent with the immunofluorescence data, EBOV and RESTV VP35 mRNA levels were similar at 1 dpi (Fig. 1A,C, andD).

The RNA-Seq data set was used to compare host gene expression profiles across samples. Statistical analysis of differential host gene expression showed significant changes in the expression of hundreds of genes in EBOV-infected MDMs at 1 and 2 dpi (Fig. 2A). In stark contrast to the strong transcriptional response to EBOV infection, only a few cellular genes were differentially expressed (DE) in RESTV-infected MDMs at these time points. The DE genes induced in RESTV-infected MDMs early and late in infection overlapped with those identified in EBOV-infected cells; about one-third of the DE genes identified in RESTV infection were also identified as DE in EBOV infection (Fig. 2B). However, the fold change in gene expression was generally less in the RESTV-infected samples than in the EBOV-infected samples, indicating a weak host response to RESTV infection. The overall gene expression pattern observed in EBOV infection was strikingly similar to that observed in LPS-treated MDMs (Fig. 2B). To better understand the functional role of genes affected by ebolavirus infection and LPS stimulation, genes identified as DE were clustered on the basis of their coexpression. This hierarchical clustering revealed four modules of gene expression (Fig. 2C). Module 1 (light blue; 572 genes) contained innate immune effector genes and genes encoding chemokines involved in immune cell recruitment (Table 1). Genes within this module were found to be profoundly upregulated in EBOV infection and LPS stimulation, whereas only a subset of these genes was impacted by RESTV infection. Similar observations were made for module 2 genes (dark blue; 1,297 genes), which are involved in antigen presentation and innate immune cellular responses (Table 1). Significant downregulation in genes involved in the epithelial tissue integrity, G-protein-coupled receptor (GPCR) signaling, and cell cycle control (grouped within module 3 (939 genes; orange) and module 4 (805 genes; red) was observed in both EBOV infection and LPS treatment (Fig. 2C;Table 1). In contrast, RESTV infection was not correlated with strong inhibition of these pathways (Fig. 2C). These data are in line with previous studies and confirm that human MDMs are strongly activated upon EBOV infection (6,9,26–30). Functional enrichment analysis was performed on global transcriptomic data using Ingenuity Pathway Analysis (IPA), an interactive software package that uses proprietary algorithms to determine functional enrichment of large, complex omics data sets such as those produced by mRNA-Seq. IPA is based on a manually curated knowledge base that links gene expression to biological functions that have been experimentally demonstrated and described in peer-reviewed articles. Functional enrichment showed a strong activation of multiple cytokine signaling pathways after EBOV infection (Fig. 3). The same overall pattern was also detected after LPS treatment, again demonstrating a striking similarity in the EBOV- and LPS-mediated responses (Fig. 3). Many of these pathways contain genes known to be involved in potent inflammatory responses such as TLR4 signaling and proinflammatory cytokine production in response to LPS. Enrichment for these pathways was not found during RESTV infection (Fig. 3). Taken together, these data suggest that RESTV fails to efficiently activate human MDMs.

RESTV infection is not associated with an IFN signature.

The EBOV VP35 and VP24 proteins counteract the host IFN response (17,18), yet the top upregulated genes in EBOV-infected MDMs at 1 dpi included type I and III IFN genes, as well as IFN-stimulated genes (ISGs) (Fig. 4A). The genes for C-X-C motif chemokine ligand 10 (CXCL10/IP10) and C-C motif chemokine ligand 5 (CCL5/RANTES), both regulated by IFN regulatory factor 3 (IRF3), were also strongly upregulated in EBOV-infected cells at 1 dpi (Fig. 4A). Similar to EBOV infection, LPS treatment led to upregulation of ISGs as well as CXCL10 and CCL5. However, we were not able to detect type I or type III IFN gene expression at any time point investigated for LPS-treated samples (Fig. 4A). In agreement with an overall lack of changes in gene expression (Fig. 2), RESTV infection led to minimal changes in the expression levels of IFN genes and ISGs (Fig. 4A).

Secretion of selected corresponding proteins was confirmed by Luminex analysis of the supernatants of infected cells. IFN-α secretion was exclusively observed in supernatants from EBOV-infected MDMs, whereas both LPS treatment and EBOV infection led to significant secretion of CXCL10 (Fig. 4B). Consistent with the RNA-Seq data, neither IFN-α nor CXCL10 was detected in the supernatants of RESTV-infected MDMs (Fig. 4B). Since it is conceivable that RESTV infection induces a delayed response at late stages of infection, we included additional time points in the Luminex analysis for two donors. In contrast to the case with EBOV, no IFN-α or CXCL10 secretion was detected in RESTV-infected MDMs up to 4 dpi (Fig. 4B, gray symbols). The lack of expression of these genes in RESTV-infected cells up to 4 dpi was confirmed by qRT-PCR analysis (data not shown). Taken together, these data show that RESTV infection of MDMs does not lead to a delayed host cell response at late stages of infection.

Expression of type I IFNs, CXCL10, and CCL5 requires the activation and nuclear translocation of the transcription factor IRF3 (reviewed in reference36). To examine whether IRF3 was activated and translocated into the nuclei of infected MDMs, we performed coimmunofluorescence analysis using antibodies directed against IRF3 and the nucleoproteins (NPs) of RESTV or EBOV. The nucleoproteins localize in large viral inclusions within the cytoplasm of the infected cells (37). In line with previous observations, viral inclusions were detected only at later time points postinfection, when viral protein expression was high (Fig. 5A) (37,38). IRF3 remained in the cytoplasm of RESTV-infected cells at all time points analyzed, whereas nuclear translocation of IRF3 was detected in a significant number of EBOV-infected cells as early as 30 min postinfection (Fig. 5). These data suggest that early events that trigger the activation of IRF3 and subsequent induction of IFN genes in EBOV-infected MDMs are absent in RESTV infection.

EBOV, but not RESTV, profoundly activates NF-κB signaling pathways.

A key regulator of cytokine and chemokine expression is nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (reviewed in reference39). We observed profound upregulation of genes involved in NF-κB signaling in EBOV-infected and LPS-treated MDMs, whereas this group of genes was only moderately affected by RESTV infection (Fig. 6A). Consistent with previous reports (6,9,26–30), robust expression of proinflammatory mediators was observed in EBOV-infected MDMs, including interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), CXCL8 (IL-8), CCL3 (MIP1α), and CCL4 (MIP1β) (Fig. 6B). In contrast, RESTV-infected MDMs remained remarkably silent. The anti-inflammatory Epstein-Barr virus-induced gene 3 (EBI3), also known as the gene for interleukin-27 subunit β (IL-27β), was the only DE cytokine gene with a >2-fold upregulation in RESTV-infected cells. A few chemokine genes were found to be upregulated, including the genes for CCL20 (MIP3α), CCL19 (MIP3β), CCL13, CXCL6, and CXCL8 (Fig. 6B). The differential expression of these genes was not unique for RESTV infection; all of them were also DE in EBOV-infected MDMs (Fig. 6B). Despite robust upregulation at the mRNA level, only moderate amounts of the proinflammatory cytokines IL-6 and TNF-α were secreted from EBOV-infected cells from some donors, compared to high levels after LPS stimulation as shown by Luminex analysis (Fig. 6C). Due to high donor variability, the observed changes in EBOV-infected cells were not statistically significant. Neither IL-6 nor TNF-α was detected in the supernatants of RESTV-infected cells at any time point analyzed. In line with the RNA data, the chemokine CXCL8 was secreted from both EBOV- and RESTV-infected MDMs (Fig. 6C). The biological relevance of CXCL8 secretion in RESTV infection remains to be determined.

An early event of canonical NF-κB activation is the nuclear translocation of the NF-κB subunit p65, which we examined by coimmunofluorescence analysis of infected or LPS-treated cells. In contrast to EBOV-infected or LPS-treated cells, nuclear accumulation of p65 was rarely observed in RESTV-infected cells (Fig. 7). At all time points analyzed, a few cells showed nuclear p65 staining in RESTV-infected samples, but this was also the case in noninfected control cells. This supports the RNA-Seq and Luminex results, which indicate that RESTV infection does not elicit a robust proinflammatory response in human MDMs. Similar to the case with IRF3, we observed nuclear translocation of p65 in EBOV-infected MDMs as early as 30 min postinfection but not at late time points (Fig. 7). These data were supported by Western blotting showing degradation of NF-κB inhibitor alpha (NFKBIA, IκBα) in EBOV-infected MDMs starting at 30 min postinfection, with a peak at 1 hpi, but not at later time points (Fig. 8A). As a control, cells were treated with LPS or TNF-α, which led to IκBα degradation as early as 15 min after treatment. IκBα prevents the nuclear translocation of NF-κB proteins, and its degradation is a crucial step in the NF-κB pathway. Controlled activation of NF-κB is achieved by a negative-feedback loop restoring IκBα levels after initial activation. The reappearance of IκBα at later time points suggests that NF-κB is not constitutively activated in EBOV-infected MDMs, which is in line with the p65 nuclear translocation data shown inFig. 7. We next performed DNA binding assays to examine if the various NF-κB subunits were able to bind to their DNA target sequences in EBOV-infected cells. MDMs were infected with EBOV, left untreated, or stimulated with LPS, and nuclear lysates were prepared at various time points. DNA binding of NF-κB subunit p65 was detected in EBOV-infected cells at 1 hpi (Fig. 8B). A similar pattern was observed for p50, although less pronounced (Fig. 8B). DNA binding of p52, c-Rel, or RelB subunits was not detected (data not shown). Together, our data show that EBOV infection of MDMs leads to early activation of canonical NF-κB signaling, whereas noncanonical signaling pathways are not activated. Compared to the case with LPS-treated samples, NF-κB activation seems to be moderate in EBOV-infected cells and only marginal in RESTV-infected MDMs.

Neither RESTV nor EBOV is able to inhibit NF-κB activation.

The lack of an inflammatory response in RESTV-infected MDMs could be due either to the lack of activating stimuli or to the inhibitory function of viral proteins that block antiviral signaling pathways, such as VP35 and VP24. To discriminate between these two possibilities, we analyzed LPS-mediated NF-κB activation at later stages of infection, when the viral proteins are expressed at high levels. MDMs were infected with RESTV or EBOV and at 1 dpi treated with LPS. LPS stimulation after RESTV infection led to significant changes in gene expression compared to RESTV-infected, untreated cells (Fig. 9A, compare RESTV versus Mock to RESTV+LPS versus RESTV) or noninfected, untreated cells (Fig. 9A, compare RESTV versus Mock to RESTV+LPS versus Mock). The gene expression profile was similar to the response to LPS alone, representing a proinflammatory signature including activation of TLR signaling and cytokine-regulated pathways (Fig. 9A, red and orange modules;Table 2). In contrast, LPS treatment after EBOV infection of MDMs resulted in minor changes in the gene expression pattern (Fig. 9A, compare EBOV versus Mock to EBOV+LPS versus EBOV) as EBOV infection alone already induced a proinflammatory response similar to LPS (Fig. 9A, EBOV versus Mock and LPS versus Mock).

As shown inFig. 7, only a minor fraction of EBOV- and RESTV-infected cells showed nuclear accumulation of p65 at 1 dpi. However, when the infected cells were treated with LPS at 1 dpi, p65 shuttled into the nucleus in almost all cells, indicating that neither RESTV nor EBOV infection interfered with LPS-induced NF-κB activation (Fig. 9B andC). This was confirmed by Luminex analysis, showing that LPS treatment of RESTV-infected cells elicited a robust cytokine and chemokine secretion (Fig. 9D). These data suggest that the lack of cytokine and chemokine induction in RESTV-infected MDMs cannot be attributed to the inhibition of immune-modulatory signaling pathways. Rather, RESTV fails to activate these pathways in human MDMs.

RESTV GP does not activate TLR4-mediated responses.

Previous studies showed that the EBOV surface protein GP is sufficient to induce a proinflammatory response via TLR4 activation (30,32,33). Since our data indicate that RESTV infection does not lead to the activation of MDMs, we next investigated if RESTV GP lacks the ability to induce a proinflammatory response. We first tested gamma-irradiated EBOV and RESTV particles for their ability to induce p65 translocation in MDMs in the absence of viral replication. p65 was translocated into the nuclei of MDMs stimulated with gamma-irradiated EBOV particles but not in cells treated with inactivated RESTV particles (Fig. 10A andB).

As mentioned above, we observed slight differences in EBOV and RESTV RNA levels at 6 hpi (Fig. 1C andD) which might be due to differences in the viral replication kinetics as reported previously (13,40,41). In addition, it has been shown recently that the particle-to-PFU ratio of EBOV stocks influenced disease outcome in an animal model (42). In order to exclude the possibility that the observed differences in the host response of MDMs to infection with EBOV or RESTV are due to differences in the replication kinetics of the two viruses or to differences in the particle-to-PFU ratio of EBOV and RESTV stocks, we next employed Ebola and Reston VLPs to determine potential differences in the ability of RESTV and EBOV GP to induce an inflammatory response in MDMs. VLPs were produced in 293T cells transfected with a mixture of expression plasmids encoding EBOV or RESTV NP, matrix protein VP40, and GP. To produce GP-free VLPs, the GP expression plasmids were omitted. The presence of EBOV and RESTV GP in VLP preparations was confirmed by Western blotting (Fig. 10C). When MDMs were stimulated with VLPs, p65 translocation was observed only in cells treated with Ebola virus VLPs (eVLPs) containing EBOV GP and not in cells treated with eVLPs lacking GP or in cells treated with Reston virus VLPs (rVLPs) with or without RESTV GP (Fig. 10D andF, bars 5, 7, 9, and 10). To confirm that the observed differences were attributed to GP and not to other VLP components, MDMs were stimulated with chimeric VLPs (eVLPs containing RESTV GP and vice versa). Nuclear p65 shuttling was observed with rVLPs containing EBOV GP but not with eVLPs containing RESTV GP, confirming that RESTV GP is not able to induce a proinflammatory response in MDMs (Fig. 10D andF, bars 8 and 11).

To examine if EBOV GP-mediated NF-κB activation could be blocked by inhibiting the TLR4 signaling pathway, MDMs were pretreated with LPS-RS, a competitive LPS antagonist fromRhodobacter sphaeroides which binds to TLR4 without inducing signaling (43,44). LPS-RS pretreatment abolished nuclear p65 translocation mediated by VLPs containing EBOV GP, indicating that the EBOV GP-triggered activation of NF-κB in MDMs is, indeed, mediated by TLR4 signaling and can be blocked by TLR4 antagonists (Fig. 10E andF, bars 5, 6, 11, and 12). Together, our data suggest that the observed differences in the proinflammatory response to EBOV and RESTV infection can be attributed to differences in GP-mediated TLR4 activation in MDMs.

Cells and viruses.

VeroE6 (ATCC CRL-1586) and 293T cells (ATCC CRL-3216) were obtained from the American Type Culture collection (ATCC). Cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (50 U/ml), and streptomycin (50 mg/ml). EBOV (Kikwit isolate, GenBank accession numberAY354458.1) and RESTV (Pennsylvania isolate, GenBank accession numberAF522874.1) stocks were grown in VeroE6 cells and purified using ultracentrifugation through a 20% sucrose cushion. Virus titers were determined in VeroE6 cells by 50% tissue culture infectious dose (TCID₅₀) assay for EBOV and focus-forming assay for RESTV. Gamma irradiation of EBOV and RESTV virus stocks was performed with a 10-megarad dose on dry ice by following standard operating procedures (SOPs) approved by the local Institutional Biosafety Committee (IBC). All work with EBOV and RESTV was performed under biosafety level 4 (BSL4) conditions at the Integrated Research Facility at the Rocky Mountain Laboratories, Division of Intramural Research, NIAID, NIH, Hamilton, MT, by following IBC-approved SOPs.

Generation of MDMs.

MDMs were generated from leukopaks (NY Biologics Inc.) or apheresed peripheral blood mononuclear cells (PBMCs; NIH Clinics Center, Department of Transfusion Medicine) using Ficoll separation (GE Healthcare) and isolation of CD14⁺ monocytes by magnetic bead selection (Milteny Biotech). A total of 1.5 × 10⁶ or 3 × 10⁵ CD14-selected monocytes were seeded into 6-well or 24-well culture plates (with or without coverslips) or at 2 × 10⁵ cells per well of a chamber slide (μ-Slide [8 well; ibidi] or Lab-Tek II chamber slide system [8 well; Nunc]) in RPMI medium without serum and allowed to adhere. Nonattached cells were removed, and RPMI medium supplemented with penicillin (50 U/ml), streptomycin (50 mg/ml), HEPES (10 mM), 5% FCS, and 5% human AB serum (Atlanta Biologicals) was added. Monocytes were incubated for 6 to 8 days to ensure differentiation into MDMs. MDMs were CD14⁺ and CD11b⁺ as confirmed by flow cytometry (data not shown). To ensure similar experimental conditions, cells from a single donor were used in a single experiment. To account for donor variations, experiments were repeated multiple times using cells obtained from different donors as noted in the figure legends. Throughout the figures, each donor is represented by a donor-specific symbol.

Generation of VLPs.

For generation of GP-containing VLPs, 3 × 10⁶ 293T cells seeded in T75 cell culture flasks were transfected with 3 μg of each expression plasmid encoding EBOV or RESTV VP40, nucleoprotein (NP), and GP using the CalPhos mammalian transfection kit (Clontech) according to the manufacturer's protocol. NP was included because it has been demonstrated to increase VLP release (73,74). The GP plasmids were replaced with 3 μg of green fluorescent protein (GFP) expression plasmid to produce VLPs lacking GP. Two days posttransfection, cell supernatants containing the VLPs were harvested and purified by ultracentrifugation through a 20% sucrose cushion. VLP protein concentration was determined using the quick-start Bradford protein assay (Bio-Rad).

Endotoxin test.

Purified, gamma-irradiated virus stocks and VLP preparations were tested for the presence of endotoxins using the Pyrogent Plus gel clotLimulus amoebocyte lysate (LAL) assay (Lonza) according to the manufacturer's protocol. Samples were found to be below the detection limit, while positive controls demonstrated that the tests were valid.

Infection and treatment of cells with viral particles, VLPs, LPS, or TNF-α.

MDMs from the same donors were infected with EBOV or RESTV at an MOI of 5 to 7. Cells were incubated at 37°C until the time of sample preparation. Equal amounts of viral particles were used for the treatment with gamma-irradiated virus. As controls, cells were left untreated or were treated with 100 ng/ml of LPS (ultrapure LPS; InvivoGen) or 20 ng/ml of TNF-α (Miltenyi Biotech) for the desired times. For VLP experiments, MDMs were treated with 1 μg/ml of VLPs for 1 h at 37°C and fixed. For LPS-RS experiments, MDMs were pretreated with 10 μg/ml of LPS-RS (ultrapure; InvivoGen) for 3 h at 37°C. Following addition of 1 ng/ml of LPS or 1 μg/ml of VLPs directly to the medium, cells were incubated for 1 h at 37°C and fixed.

RNA sample preparation.

Total RNA from 1.5 × 10⁶ (6-well format) or 3 × 10⁵ (24-well format) MDMs was isolated at the desired time points using TRIzol reagent (Invitrogen) according to the manufacturer's protocol and IBC-approved SOPs. RNA concentration was determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific). For VP35 mRNA quantification by qRT-PCR, mRNA was purified from total RNA using the Poly(A)Purist kit (Ambion) according to the manufacturer's protocol.

Library preparation, RNA sequencing, and processing.

Libraries for mRNA sequencing were constructed using the Kapa stranded mRNA-Seq kit (Kapa Biosystems) using RNA samples from 3 separate donors. Libraries were quality controlled and quantified using the BioAnalyzer 2100 (Agilent Technologies, Inc.) and QuBit (Invitrogen) systems. Libraries were clonally amplified and sequenced on an Illumina NextSeq 500 to achieve a target density of approximately 200,000 to 220,000 clusters/mm² on the flow cell with dual indexed paired-end sequencing at a 76-bp length using NextSeq 500 NCS v1.3 software. Raw reads (76 bp) had their adaptor sequences removed. General quality control of the raw reads was performed using FastQC. rRNA reads were removed via mapping by Bowtie (v2.1.0) using an index of human, mouse, and rat rRNA sequences (75). On average ∼10% of the reads from each sample were identified as mapping to rRNA and were removed from downstream analysis. Reads were also aligned to the genomes of the EBOV (Kikwit isolate, GenBank accession numberAY354458.1) and RESTV (Pennsylvania isolate, GenBank accession numberAF522874.1) using Bowtie2. Additionally, reads mapping to VP35 regions in either genome were counted using HTseq-count. Following removal of viral reads, the remaining reads were then mapped against a human reference genome (hg19, build GRCh37, from the UCSC genome browser using STAR [v2.4.0h1]) (76). Quantitative gene counts were produced from this alignment using the Python package HT-Seq utilizing the human annotation associated with the genome (77).

Differential expression analysis.

Normalization and differential expression analysis were carried out using R (v3.1.3) and the software package edgeR (v3.10.2) (78). Prior to differential analysis expression, genes with no counts were removed and counts across samples were normalized using the weighted trimmed mean of M-values. Remaining genes without at least three samples with counts were additionally removed leaving 14,288 genes with an average of ∼10 M reads per sample. Differentially expressed genes were determined between treatments relative to time-matched and donor-matched samples using a generalized linear model implemented in edgeR. Cutoffs for differential gene expression were defined as an absolute fold change of 2 and aP value of ≤0.05 after false-discovery rate adjustment using the Benjamini-Hochberg multiple-testing correction (79).

Functional enrichment analysis.

Functional analysis of DE genes was done using Ingenuity Pathway Analysis (IPA; Ingenuity Systems). IPA assesses enrichment of biological pathways and functional categories using a proprietary, manually curated knowledgebase derived from peer-reviewed published studies. IPA functional enrichment was calculated using a right-tailed Fisher exact test with a threshold of significance set at aP value of 0.05. Pathway enrichment is reported as (−log₁₀P value) as determined by the Fisher exact test. Additional functional gene annotation was obtained from the human gene database, GeneCards (http://www.genecards.org) (v4.0.5) (80).

qRT-PCR analysis.

Five nanograms of mRNA was analyzed by quantitative reverse transcription-PCR (qRT-PCR) using the QuantiFast SYBR green RT-PCR kit (Qiagen). qRT-PCR-based quantification of VP35 mRNA was performed using a primer pair that binds to both the EBOV and RESTV VP35 genes without mismatches. Validated QuantiTect primer assays (Qiagen) for β2-microglobulin were used as an endogenous control for normalization. qRT-PCR was performed in a CFX96 real-time PCR cycler (Bio-Rad). Fold expression levels compared to those of noninfected samples collected at each corresponding time point were quantified using the threshold cycle (ΔΔC_T) method (Bio-Rad CFX Manager 1.5 software). Statistical analysis was performed with a two-tailedt test using GraphPad Prism 5 software.

Luminex analysis.

At the desired time points after infection or treatment, 1 ml of supernatant was collected from 1.5 × 10⁶ (6-well format) or 3 × 10⁵ (24-well format) MDMs. For inactivation, samples were exposed to 10 megarads of gamma irradiation on dry ice by following IBC-approved SOPs. Samples were clarified by low-speed centrifugation and analyzed using the Procarta human cytokine assay (Affymetrix). Mean fluorescence intensity was measured to calculate final concentration in pictograms per milliliter using Bioplex200 and Bioplex Manager 5 software (Bio-Rad). Statistical analysis of data was performed using one-way analysis of variance (ANOVA) using GraphPad Prism 5 software.

Immunofluorescence analysis.

A total of 2 × 10⁵ MDMs seeded per well of μ-Slide (ibidi) or Lab-Tek II (Nunc) 8-well chamber slides or 3 × 10⁵ MDMs seeded on coverslips were infected or treated as described above. At the desired time points, cells were fixed with 4% paraformaldehyde overnight and removed from the BSL4 facility by following IBC-approved SOPs. Cells were permeabilized with acetone-methanol (1:1) at −20°C for 10 min. Primary antibodies include a mouse anti-EBOV NP antibody (gift from G. Olinger, formerly USAMRIID, Virology Division, Frederick, MD), a mouse anti-RESTV NP antibody, and a rabbit anti-IRF3 antibody and a rabbit anti-p65 antibody (both Santa Cruz). Secondary antibodies were conjugated with Alexa Fluor 495 or Alexa Fluor 488 (Molecular Probes). 4′,6-Diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) was used for nucleus staining. At least 100 cells per sample were counted to determine infection rates or percentage of cells with nuclear localization of p65 or IRF3. GraphPad Prism 5 software was used to calculate standard errors of the means (SEM) and to perform statistical analysis of data using one-way ANOVA for p65 and IRF3 localization.

Western blot analysis.

At the desired time points, 1.5 × 10⁶ (6-well format) MDMs were scraped into phosphate-buffered saline (PBS) and pelleted by low-speed centrifugation. Whole-cell extracts were prepared in cell extraction buffer (Biosource) complemented with protease inhibitor mix (complete; Roche) and serine/threonine phosphatase inhibitor (Calyculin A; CST) according to the manufacturer's protocol. Cell lysates were transferred to fresh tubes containing 2× SDS sample buffer. Inactivation of EBOV or RESTV and corresponding controls was performed by incubating the samples at 95°C for 10 min by following IBC-approved SOPs. Samples were subjected to Western blot analysis using antibodies against IκBα (Santa Cruz) and β-actin (Abcam). Secondary antibodies (Dianova) were conjugated with horseradish peroxidase. Detection was performed using SuperSignal chemiluminescent substrate (Pierce/Thermo Scientific) according to the manufacturer's protocol. For VLP analysis, an aliquot of each VLP preparation was mixed with 2× SDS sample buffer and subjected to Western blot analysis using antibodies against EBOV GP1, RESTV GP1 (IBT Bioservices), and EBOV VP40. The EBOV VP40 antibody cross-reacts with RESTV VP40. The secondary antibody was conjugated to IRDye800 (Li-Cor), and protein bands were visualized using the Odyssey imaging system and software (Li-Cor).

DNA binding assay.

At the desired time points, nuclear lysates from 6 × 10⁶ MDMs were prepared using the NucBuster protein extraction kit (Novagen). Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce/Thermo Scientific), and 5 μg of protein per sample was analyzed using the TransAM NF-κB family kit assay (Active Motif) according to the manufacturer's instructions. Statistical analysis of data was performed using one-way ANOVA with GraphPad Prism 5 software.

Accession number(s).

Raw and normalized sequencing data files and differentially expressed genes were deposited in the public Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) under accession numberGSE84188.

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