Figure 1.
NLRP3 inflammasome activation by EMCV.
(A) BMMs were infected with EMCV or influenza virus PR8 in the presence or absence of LPS at an MOI of 1.5. (B) LPS-primed BMMs were infected with EMCV at the indicated MOIs. (C) Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup) and pro-IL-1β in extracts (Cell) of LPS-primed BMDCs infected with EMCV. (D) LPS-primed BMDCs were treated with EMCV, influenza virus PR8 or ATP in the presence or absence of yVAD-CHO. (E) LPS-primed RAW264.7 cells stably expressing shRNA against NLRP3, ASC, caspase-1, or EGFP mRNAs were infected with EMCV or influenza virus PR8. Cell-free supernatants were collected at 24 h after infection and analyzed for IL-1β or IL-6 by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± S.D. (A, B, D, and E). **, P<0.01; ***, P<0.001.
Figure 2.
Viral RNA-independent recognition of EMCV by the NLRP3 inflammasome.
(A) LPS-primed BMDCs were inoculated with live or UV-inactivated EMCV. (B) BMDCs were primed with LPS for 3 h, and then treated with 100 µM of cycloheximide (CHX) for 1 h prior to the treatment with ATP or infection with EMCV. (C, D) BMMs were infected with EMCV or transfected with 10 µg/ml poly(dA∶dT) or various amounts (12.5, 25, 50 µg) of total RNA from uninfected cells (uRNA), total RNA from EMCV-infected cells (iRNA), or EMCV genomic RNA (vRNA) for 18 h. Total RNA was extracted from virus-infected or RNA-transfected BMMs. IFN-β mRNA levels were assessed by RT quantitative PCR. GAPDH was used as an internal control. Data are pooled from three independent experiments (C). Supernatants were analyzed for the presence of mature IL-1β by ELISA (D). (E) Viral genomic RNAs were extracted from indicated amounts of EMCV, and their concentrations were determined. (F) Virus titers in the supernatants of the cells examined in (D) are shown. Data are representative of at least three independent experiments, and indicate the mean ± S.D. N.S., not significant. **, P<0.01.
Figure 3.
NLRP3 inflammasome activation by EMCV viroporin 2B.
(A) LPS-primed BMMs were infected with the lentivirus expressing EMCV 2A, 2B, or 2C, or GFP. Supernatants were collected at 24 h post infection and analyzed for IL-1β by ELISA. (B) HEK293T cells were transfected with the expression plasmid encoding Flag- or HA-tagged EMCV 2A, 2B, or 2C, or NLRP3 or empty vector. Samples were analyzed by immunoblot with rabbit polyclonal antibody against Flag or mouse monoclonal antibody against HA. (C) HeLa cells were transfected with the expression plasmid encoding Flag-tagged EMCV 2A, 2B, or 2C (green) and that encoding either pDsRed-monomer-Golgi or pDsRed2-ER (red), and observed with a confocal microscope at 24 h after transfection. (D) LPS-primed BMMs were stimulated with EMCV or nigercin for 24 h in the presence or absence of yVAD-CHO. Cells were stained with anti-NLRP3 (green) and anti-GM130 (Golgi marker) or anti-calnexin (ER marker) (red) and analyzed by a confocal microscopy. Mock-treated cells were also examined. Nuclei were visualized by staining with DAPI. (E) HeLa cells were transfected with the expression plasmid encoding Flag-tagged NLRP3 (green) and that encoding HA-tagged EMCV 2A, 2B, or 2C, or influenza virus M2 protein (red). Nuclei were visualized by staining with DAPI. Scale bars represent 10 µm. Data are representative of at least three independent experiments, and indicate the mean ± S.D (A). ***, P<0.001.
Figure 4.
Requirement of increased intracellular Ca2+ concentration for NLRP3 inflammasome activation.
(A) Intracellular Ca2+ concentration in HeLa cells infected with EMCV. The ratio of fluorescent intensities was recorded with an ICCD camera/image analysis system. (B) LPS-primed BMDCs were infected with EMCV. (C, D) LPS-primed BMMs were stimulated with thapsigarsin (TG) or ionomycin. (D) Cells were stained with anti-NLRP3 (green) and anti-GM130 or anti-calnexin (red) and analyzed by a confocal microscopy. Nuclei were visualized by staining with DAPI. (E) LPS-primed BMMs were infected with EMCV or L. monocytogenes in the presence or absence of EGTA. (F) LPS-primed BMMs were infected with EMCV in the presence or absence of BAPTA-AM. LDH activity was measured as control for cytotoxicity. (G–H) HeLa cells were transfected with 2.5 µg each of pCA7-Flag-2A, pCA7-Flag-2B, or pCA7-Flag-2C and 0.5 µg of pDsRed2-ER/Golgi-G-GECO1, which encodes the EGFP chimera that is targeted to ER/Golgi and changes its fluorescence levels according to the Ca2+ concentrations, and observed under a phase-contrast or fluorescence microscope at 72 h after transfection (G). At 72 h after transfection, samples were analyzed by immunoblot with mouse monoclonal antibody against EGFP or mouse monoclonal antibody against actin (H). Cell-free supernatants were collected at 24 h after stimulation and analyzed for IL-1α or IL-1β by ELISA (B–C, E–F). Data are representative of at least three independent experiments, and indicate the mean ± S.D. (B–C, E–F). *, P<0.05; **, P<0.01; ***, P<0.001.
Figure 5.
Mitochondrial ROS- and cathepsin B-independent activation of NLRP3 inflammasome by EMCV.
(A) LPS-primed BMMs were infected with EMCV. Cell-free supernatants were collected at indicated time points and analyzed for IL-1β by ELISA (left y axis, filled bars). Cells were collected at indicated time points and stained with MitoSOX for 30 min and analyzed by flow cytometry. The proportions of ROS-producing mitochondria are shown (right y axis, open circles). (B–C) LPS-primed BMMs were treated with EMCV, ATP, MSU (B), or Alum (C) in the presence or absence of Mito-TEMPO (500 µM), a scavenger specific for mitochondrial ROS, or cathepsin B inhibitor CA-074 Me (10 µM) (C). Cell-free supernatants were collected at 24 h (EMCV, Alum) or 6 h (ATP) post infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± S.D. *, P<0.05.
Figure 6.
A schematic model of EMCV-induced IL-1β secretion.
After infection, the newly synthesized viral polyprotein is cleaved by viral proteases. Within the Golgi apparatus or other cytoplasmic structures, EMCV 2B protein (red) induces Ca2+ flux and stimulates the NLRP3 inflammasome pathway. The activated caspase-1 catalyzes proteolytic processing of pro-IL-1β into its mature form, leading to their secretion across the plasma membrane.