Severe Acute Respiratory Syndrome Coronavirus Viroporin 3a Activates the NLRP3 Inflammasome

Abstract

Nod-like receptor family, pyrin domain-containing 3 (NLRP3) regulates the secretion of proinflammatory cytokines interleukin 1 beta (IL-1β) and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus (EMCV) 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus (SARS-CoV) activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K⁺ efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation.

Keywords: IL-1β; SARS-CoV; inflammasome; inflammation; viroporin.

FIGURE 1

The 3a protein of SARS-CoV stimulates IL-1β secretion. (A–C) HEK293FT cells were transfected with pLenti6-E-V5, pLenti6-3a-V5, pLenti6-M-V5 (A), pLenti-GFP-V5 (B), or pLenti-M2-V5 plasmids (C). Samples were analyzed by immunoblot with mouse monoclonal antibodies against V5-tag (A), GFP (B), or influenza virus M2 (C). (D,E) LPS-primed BMM were infected with the lentivirus expressing SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B at MOI 0.25 (D) or 0.1 (E). Supernatants were collected at 24 h post-infection and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD (D,E); ^∗∗∗P < 0.001.

FIGURE 2

Ion channel activity of the 3a protein is required for IL-1β secretion. (A) SARS-CoV 3a protein; below, amino acid sequence of cysteine-rich domain (residue 127–133) of wild-type 3a and 3a-CS mutant. (B) LPS-primed BMM were infected with the lentivirus expressing SARS-CoV E, V25F, 3a, 3a-CS, or M at MOI 0.25. Supernatants were collected at 24 h post-infection and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD (B); ^∗∗∗P < 0.001.

FIGURE 3

Subcellular localization of SARS-CoV 3a protein and 3a-CS mutant. (A,B) HeLa cells were transfected with the expression plasmid encoding flag-tagged 3a or 3a-CS and that encoding either DsRed-monomer-Golgi (A) or ER-mCherry (B), and observed with a confocal microscope at 24 h post-transfection. Scale bars, 10 μm. Data are representative of at least three independent experiments.

FIGURE 4

NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 μm. Data are representative of at least three independent experiments.

FIGURE 5

K⁺ efflux is required for activation of the NLRP3 inflammasome by SARS-CoV 3a protein. (A,B) BMMs were infected with influenza virus A/PR8 (A) or lentiviruses expressing SARS-CoV 3a or E proteins (B) and cultured in the presence or absence of KCl (130 mM). Cell-free supernatants were collected at 24 h post-infection, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; ^∗∗P < 0.01 and ^∗∗∗P < 0.001.

FIGURE 6

Mitochondrial ROS-dependent activation of the NLRP3 inflammasome by SARS-CoV 3a protein. (A,B) LPS-primed BMMs were stimulated with ATP (A) or lentiviruses expressing SARS-CoV 3a or E proteins (B) in the presence or absence of Mito-TEMPO (500 μM). Cell-free supernatants were collected at 24 h (lentiviruses) 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 ± SD; ^∗∗P < 0.01 and ^∗∗∗P < 0.001.