Fig 2: EBV-encoded miRNAs regulate the expression of cellular genes and proteins involved in type I IFN activation. (A to E) 293T cells were cotransfected with the indicated wild-type luciferase reporter plasmids (WT) for RIG-I/DDX58 (A), IRAK2 (B), Viperin/RSAD2 (C), FYN (D), and OAS2 (E) or reporter plasmid in which the predicted seed sequence was mutated (mut) together with or without an miRNA-encoding plasmid. The luciferase expression in these cells was assessed and normalized to lysates from cells cotransfected with the wild-type 3'-UTR reporter and empty miRNA expression plasmid (Ø). P values were calculated using the one-way analysis of variance (ANOVA) test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (F) Human primary B lymphocytes were infected with wild-type EBV (r_wt/B95.8) or EBV devoid of its miRNAs (r_?miR). Five days postinfection, EBV-infected cells were counted and seeded at the same density. Four days later, the cells were lysed and the lysates were subjected to quantitative Western blot analysis using the Western blot stain-free TGX Bio-Rad normalization approach (Bio-Rad). Blots were probed with antibodies against IPO7, Viperin/RSAD2, TLR9, TLR7, FYN, RIG-I/DDX58, and OAS2. Protein levels were quantified and used to normalize the levels of the specific protein signals. Ratios of protein levels in cells infected with wild-type virus versus cells infected with mutant virus are shown. Reported are the results of four independent biological replicates.

Fig 3: Inhibitory effect of TLR9 siRNA on MWCNT-induced 8-nitroG formation. a Immunofluorescent images of reduced TLR9 expression and the absence of 8-nitroG formation in siRNA-transfected cells. A549 cells were transfected with negative control or TLR9 siRNA (siRNA-1 and siRNA-2). TLR9 expression and 8-nitroG formation were examined by fluorescent immunocytochemistry. Hoechst, Hoechst 33258. Magnification, X400. b Relative staining intensity of TLR9 in siRNA-transfected A549 cells. The staining intensity of TLR9 per cell was analyzed by an ImageJ software. Relative staining intensity of the control was set at 1. Data represent means ± SD of 4 independent experiments. ***p < 0.001, compared with the control; ### p < 0.001, compared with negative control siRNA-transfected cells. c Reduction of TLR9 expression in siRNA-transfected cells. A549 cells transfected with TLR9 siRNA were lysed and treated, and then Western blotting was performed to detect TLR9 and GAPDH as described in Methods. d Relative intensity of TLR9 expression in siRNA-transfected A549 cells. The relative band intensity of TLR9 normalized by GAPDH was measured with an ImageJ software. Data represent means ± SD of 3 independent experiments. **p < 0.01 and ***p < 0.001, compared with the control; # p < 0.05 and ## p < 0.01, compared with negative control siRNA-transfected cells. e Inhibitory effect of TLR9 siRNA on MWCNT-induced 8-nitroG formation. After siRNA transfection, A549 cells were treated with 1 µg/ml CNT-L for 8 h. 8-NitroG formation was analyzed by fluorescent immunocytochemistry. Magnification, X200. f Quantitative image analysis for effects of TLR9 siRNA on MWCNT-induced 8-nitroG formation. Staining intensity was analyzed as described in (b). Data represent means ± SD of 3-4 independent experiments. ***p < 0.001, compared with the control, ### p < 0.001, compared with MWCNT alone, $$ p < 0.01 and $$$ p < 0.001, compared with MWCNT + negative control siRNA. g Effect of TLR9 siRNA on NO release from MWCNT-exposed cells. After siRNA transfection, A549 cells were treated with 1 µg/ml CNT-L for 4 h. Data represent means ± SD of 5-6 independent experiments. ***p < 0.001, compared with the control; # p < 0.05 and ### p < 0.001, compared with CNT-L-treated cells. Statistical analysis was performed by ANOVA followed by Tukey’s test

Fig 4: EBV infects a fraction of pDCs, which are the main producer of IFN-a. (A) Schematic overview of the EBV virion-based ß-lactamase (BlaM) fusion assay. Expression plasmids encoding BZLF1, BALF4, and the gp350:ß-lactamase (BlaM) fusion protein were cotransfected into EBV producer cells (HEK293 wt/B95.8 [2089]; Table S1). Target cells were infected with the gp350:BlaM assembled virions for 4 h and were subsequently loaded with the CCF4-AM substrate. If ß-lactamase reaches the cytoplasm of the target cells, it cleaves the ß-lactam ring of CCF4, which leads to a detectable shift in emission wavelength from 520 nm to 447 nm. Below, a schematic workflow of the steps of the experiment performed in panel C is shown. PBMCs were prepared from human buffy coats, and pDCs were isolated via negative selection. PBMCs and pDCs were infected with gp350:BlaM-assembled wt/B95.8 (2089) EBV at an MOI of 1. As negative controls, the cells were infected with equivalent doses of unmodified wt/B95.8 (2089) EBV. All cells were cultivated for 4 h before they were loaded with CCF4-AM and incubated at room temperature for 20 h. In the case of PBMCs, the cells were stained for B cells using a CD19 antibody, and in the case of isolated pDCs, the cells were stained with two CD303- and CD304-specific antibodies. The percentage of B cells and pDCs with green (uncleaved CCF4 substrate) and blue (cleaved CCF4 substrate) fluorescence was determined using flow cytometry. (B) Schematic representation of the steps of the experiment performed in panel D. pDCs were isolated via negative selection out of PBMCs gained from buffy coats. pDCs were treated with EBV wt/B95.8 (2089) or EBV wt/B95.8 (2089) gp350:BlaM for 4 h. Cells were washed and loaded with CCF4-AM at room temperature for 20 h. The pDCs were sorted for green and blue cell populations, and the same cell numbers were plated on 96-well cluster plates. The cells were incubated at 37°C for 20 h, and the supernatants were collected. The IFN-a levels were quantified by ELISA. (C) Results of experiments with B cells and pDCs as detailed for panel A. The y axis shows the fraction of cells with blue fluorescence after cleavage of CCF4. Controls are cells infected with unmodified wt/B95.8 (2089) EBV. (D) Results of experiments with isolated pDCs obtained from buffy coats as described for panel B. A total of 2 × 104 pDCs that had been sorted according to their blue fluorescence release close to 1 ng/ml IFN-a, whereas the same number of pDCs that did not show a fluorescence shift in the emission wavelength released very little IFN-a. As negative control, cells were left uninfected. As a positive control, cells that were infected but did not show a fluorescence shift were stimulated with the TLR9 agonist ODN2216.

Fig 5: EBV-induced IFN-a release by PBMCs depends on viral DNA and TLR9 signaling. (A) PBMCs were infected with r_?miR EBV or with virus-like particles (VLP Cre5), which do not contain viral DNA. Noninfected (NI) cells and cells treated with the TRL9 agonist ODN2216 served as negative and positive controls, respectively. The culture supernatants were collected 20 h postinfection, and the IFN-a levels were quantitated by ELISA. Means and standard deviations from three to four biological replicates are shown. (B) PBMCs were infected with EBV r_wt/B95.8 at different MOIs as indicated, with or without treatment with 100 µM chloroquine. The culture supernatants were collected 20 h postinfection, and the IFN-a levels were quantitated by ELISA. (C) PBMCs were infected with EBV r_?miR using different MOIs as indicated. The cells were left untreated or were treated with 0.25 µM TLR9 antagonist ODN2088 or 0.25 µM ODN2088 control oligonucleotide, termed ODN2087. ODN2087 acts as a TLR9 antagonist control. Cells treated with different concentrations of the TRL9 agonist ODN2216 served as positive controls, and noninfected (NI) cells act as a background control. The culture supernatants were collected 20 h postinfection, and the IFN-a levels were quantitated by ELISA. Reported are the results of four independent biological replicates. ODN2216 is a TRL-9 agonist that was used a positive control.