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

I-Yin Chen, Miyu Moriyama, Ming-Fu Chang, Takeshi Ichinohe, I-Yin Chen, Miyu Moriyama, Ming-Fu Chang, Takeshi Ichinohe

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.

Figures

FIGURE 1
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
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
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
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
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
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.

References

    1. Allen I. C., Scull M. A., Moore C. B., Holl E. K., Mcelvania-Tekippe E., Taxman D. J., et al. (2009). The NLRP3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral RNA. Immunity 30 556–565. 10.1016/j.immuni.2009.02.005
    1. Arlehamn C. S., Petrilli V., Gross O., Tschopp J., Evans T. J. (2010). The role of potassium in inflammasome activation by bacteria. J. Biol. Chem. 285 10508–10518. 10.1074/jbc.M109.067298
    1. Bauernfeind F., Ablasser A., Bartok E., Kim S., Schmid-Burgk J., Cavlar T., et al. (2011). Inflammasomes: current understanding and open questions. Cell Mol. Life Sci. 68 765–783. 10.1007/s00018-010-0567-4
    1. Castano-Rodriguez C., Honrubia J. M., Gutierrez-Alvarez J., Dediego M. L., Nieto-Torres J. L., Jimenez-Guardeno J. M., et al. (2018). Role of severe acute respiratory syndrome Coronavirus viroporins E, 3a, and 8a in replication and pathogenesis. mBio 9:e2325–17. 10.1128/mBio.02325-17
    1. Chakrabarti A., Banerjee S., Franchi L., Loo Y. M., Gale M., Jr., Núñez G., et al. (2015). RNase L activates the NLRP3 inflammasome during viral infections. Cell Host Microbe 17 466–477. 10.1016/j.chom.2015.02.010
    1. Chan C. M., Tsoi H., Chan W. M., Zhai S., Wong C. O., Yao X., et al. (2009). The ion channel activity of the SARS-coronavirus 3a protein is linked to its pro-apoptotic function. Int. J. Biochem. Cell Biol. 41 2232–2239. 10.1016/j.biocel.2009.04.019
    1. Chen I. Y., Ichinohe T. (2015). Response of host inflammasomes to viral infection. Trends Microbiol. 23 55–63. 10.1016/j.tim.2014.09.007
    1. Chen W., Xu Y., Li H., Tao W., Xiang Y., Huang B., et al. (2014). HCV genomic RNA activates the NLRP3 inflammasome in human myeloid cells. PLoS One 9:e84953. 10.1371/journal.pone.0084953
    1. Drosten C., Gunther S., Preiser W., Van Der Werf S., Brodt H. R., Becker S., et al. (2003). Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med. 348 1967–1976. 10.1056/NEJMoa030747
    1. Duewell P., Kono H., Rayner K. J., Sirois C. M., Vladimer G., Bauernfeind F. G., et al. (2010). NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 464 1357–1361. 10.1038/nature08938
    1. Farag N. S., Breitinger U., El-Azizi M., Breitinger H. G. (2017). The p7 viroporin of the hepatitis C virus contributes to liver inflammation by stimulating production of Interleukin-1beta. Biochim. Biophys. Acta Mol. Basis Dis. 1863 712–720. 10.1016/j.bbadis.2016.12.006
    1. Fernandes-Alnemri T., Wu J., Yu J. W., Datta P., Miller B., Jankowski W., et al. (2007). The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation. Cell Death Differ. 14 1590–1604. 10.1038/sj.cdd.4402194
    1. Fouchier R. A., Kuiken T., Schutten M., Van Amerongen G., Van Doornum G. J., Van Den Hoogen B. G., et al. (2003). Aetiology: Koch’s postulates fulfilled for SARS virus. Nature 423:240. 10.1038/423240a
    1. Halle A., Hornung V., Petzold G. C., Stewart C. R., Monks B. G., Reinheckel T., et al. (2008). The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nat. Immunol. 9 857–865. 10.1038/ni.1636
    1. He L., Ding Y., Zhang Q., Che X., He Y., Shen H., et al. (2006). Expression of elevated levels of pro-inflammatory cytokines in SARS-CoV-infected ACE2+ cells in SARS patients: relation to the acute lung injury and pathogenesis of SARS. J. Pathol. 210 288–297. 10.1002/path.2067
    1. Hornung V., Bauernfeind F., Halle A., Samstad E. O., Kono H., Rock K. L., et al. (2008). Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat. Immunol. 9 847–856. 10.1038/ni.1631
    1. Hornung V., Latz E. (2010). Critical functions of priming and lysosomal damage for NLRP3 activation. Eur. J. Immunol. 40 620–623. 10.1002/eji.200940185
    1. Ichinohe T., Lee H. K., Ogura Y., Flavell R., Iwasaki A. (2009). Inflammasome recognition of influenza virus is essential for adaptive immune responses. J. Exp. Med. 206 79–87. 10.1084/jem.20081667
    1. Ichinohe T., Pang I. K., Iwasaki A. (2010). Influenza virus activates inflammasomes via its intracellular M2 ion channel. Nat. Immunol. 11 404–410. 10.1038/ni.1861
    1. Ichinohe T., Yamazaki T., Koshiba T., Yanagi Y. (2013). Mitochondrial protein mitofusin 2 is required for NLRP3 inflammasome activation after RNA virus infection. Proc. Natl. Acad. Sci. U.S.A. 110 17963–17968. 10.1073/pnas.1312571110
    1. Ito M., Yanagi Y., Ichinohe T. (2012). Encephalomyocarditis virus viroporin 2B activates NLRP3 inflammasome. PLoS Pathog. 8:e1002857. 10.1371/journal.ppat.1002857
    1. Jiang J., Stoyanovsky D. A., Belikova N. A., Tyurina Y. Y., Zhao Q., Tungekar M. A., et al. (2009). A mitochondria-targeted triphenylphosphonium-conjugated nitroxide functions as a radioprotector/mitigator. Radiat. Res. 172 706–717. 10.1667/RR1729.1
    1. Johnson K. E., Chikoti L., Chandran B. (2013). Herpes simplex virus 1 infection induces activation and subsequent inhibition of the IFI16 and NLRP3 inflammasomes. J. Virol. 87 5005–5018. 10.1128/JVI.00082-13
    1. Kawai T., Akira S. (2010). The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11 373–384. 10.1038/ni.1863
    1. Kayagaki N., Stowe I. B., Lee B. L., O’rourke K., Anderson K., Warming S., et al. (2015). Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526 666–671. 10.1038/nature15541
    1. Ksiazek T. G., Erdman D., Goldsmith C. S., Zaki S. R., Peret T., Emery S., et al. (2003). A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348 1953–1966. 10.1056/NEJMoa030781
    1. Kuiken T., Fouchier R. A., Schutten M., Rimmelzwaan G. F., Van Amerongen G., Van Riel D., et al. (2003). Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 362 263–270. 10.1016/S0140-6736(03)13967-0
    1. Lu W., Zheng B. J., Xu K., Schwarz W., Du L., Wong C. K., et al. (2006). Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release. Proc. Natl. Acad. Sci. U.S.A. 103 12540–12545. 10.1073/pnas.0605402103
    1. Mariathasan S., Weiss D. S., Newton K., Mcbride J., O’rourke K., Roose-Girma M., et al. (2006). Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440 228–232. 10.1038/nature04515
    1. Matsuyama S., Ujike M., Morikawa S., Tashiro M., Taguchi F. (2005). Protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection. Proc. Natl. Acad. Sci. U.S.A. 102 12543–12547. 10.1073/pnas.0503203102
    1. Medzhitov R. (2001). Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1 135–145. 10.1038/35100529
    1. Minakshi R., Padhan K. (2014). The YXXPhi motif within the severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein is crucial for its intracellular transport. Virol. J. 11:75. 10.1186/1743-422X-11-75
    1. Mitoma H., Hanabuchi S., Kim T., Bao M., Zhang Z., Sugimoto N., et al. (2013). The DHX33 RNA helicase senses cytosolic RNA and activates the NLRP3 inflammasome. Immunity 39 123–135. 10.1016/j.immuni.2013.07.001
    1. Moriyama M., Chen I. Y., Kawaguchi A., Koshiba T., Nagata K., Takeyama H., et al. (2016). The RNA- and TRIM25-binding domains of influenza virus NS1 protein are essential for suppression of NLRP3 inflammasome-mediated IL-1beta secretion. J. Virol. 90 4105–4114. 10.1128/JVI.00120-16
    1. Munoz-Planillo R., Kuffa P., Martinez-Colon G., Smith B. L., Rajendiran T. M., Nunez G. (2013). K(+) efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity 38 1142–1153. 10.1016/j.immuni.2013.05.016
    1. Murakami T., Ockinger J., Yu J., Byles V., Mccoll A., Hofer A. M., et al. (2012). Critical role for calcium mobilization in activation of the NLRP3 inflammasome. Proc. Natl. Acad. Sci. U.S.A. 109 11282–11287. 10.1073/pnas.1117765109
    1. Nakahira K., Haspel J. A., Rathinam V. A., Lee S. J., Dolinay T., Lam H. C., et al. (2011). Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat. Immunol. 12 222–230. 10.1038/ni.1980
    1. Negash A. A., Ramos H. J., Crochet N., Lau D. T., Doehle B., Papic N., et al. (2013). IL-1beta production through the NLRP3 inflammasome by hepatic macrophages links hepatitis C virus infection with liver inflammation and disease. PLoS Pathog. 9:e1003330. 10.1371/journal.ppat.1003330
    1. Nieto-Torres J. L., Verdia-Baguena C., Jimenez-Guardeno J. M., Regla-Nava J. A., Castano-Rodriguez C., Fernandez-Delgado R., et al. (2015). Severe acute respiratory syndrome coronavirus E protein transports calcium ions and activates the NLRP3 inflammasome. Virology 485 330–339. 10.1016/j.virol.2015.08.010
    1. Pang I. K., Ichinohe T., Iwasaki A. (2013). IL-1R signaling in dendritic cells replaces pattern-recognition receptors in promoting CD8(+) T cell responses to influenza a virus. Nat. Immunol. 14 246–253. 10.1038/ni.2514
    1. Parthasarathy K., Ng L., Lin X., Liu D. X., Pervushin K., Gong X., et al. (2008). Structural flexibility of the pentameric SARS coronavirus envelope protein ion channel. Biophys. J. 95 L39–L41. 10.1529/biophysj.108.133041
    1. Peiris J. S., Lai S. T., Poon L. L., Guan Y., Yam L. Y., Lim W., et al. (2003). Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361 1319–1325. 10.1016/S0140-6736(03)13077-2
    1. Perlman S., Dandekar A. A. (2005). Immunopathogenesis of coronavirus infections: implications for SARS. Nat. Rev. Immunol. 5 917–927. 10.1038/nri1732
    1. Pervushin K., Tan E., Parthasarathy K., Lin X., Jiang F. L., Yu D., et al. (2009). Structure and inhibition of the SARS coronavirus envelope protein ion channel. PLoS Pathog. 5:e1000511. 10.1371/journal.ppat.1000511
    1. Petrilli V., Papin S., Dostert C., Mayor A., Martinon F., Tschopp J. (2007). Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration. Cell Death Differ. 14 1583–1589. 10.1038/sj.cdd.4402195
    1. Schroder K., Zhou R., Tschopp J. (2010). The NLRP3 inflammasome: a sensor for metabolic danger? Science 327 296–300. 10.1126/science.1184003
    1. Shi J., Zhao Y., Wang K., Shi X., Wang Y., Huang H., et al. (2015). Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526 660–665. 10.1038/nature15514
    1. Shimada K., Crother T. R., Karlin J., Dagvadorj J., Chiba N., Chen S., et al. (2012). Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis. Immunity 36 401–414. 10.1016/j.immuni.2012.01.009
    1. Subramanian N., Natarajan K., Clatworthy M. R., Wang Z., Germain R. N. (2013). The adaptor MAVS promotes NLRP3 mitochondrial localization and inflammasome activation. Cell 153 348–361. 10.1016/j.cell.2013.02.054
    1. Tan Y. J., Lim S. G., Hong W. (2005). Characterization of viral proteins encoded by the SARS-coronavirus genome. Antiviral Res. 65 69–78. 10.1016/j.antiviral.2004.10.001
    1. Torres J., Maheswari U., Parthasarathy K., Ng L., Liu D. X., Gong X. (2007). Conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of SARS coronavirus envelope protein. Protein Sci. 16 2065–2071. 10.1110/ps.062730007
    1. Triantafilou K., Kar S., Van Kuppeveld F. J., Triantafilou M. (2013). Rhinovirus-induced calcium flux triggers NLRP3 and NLRC5 activation in bronchial cells. Am. J. Respir. Cell Mol. Biol. 49 923–934. 10.1165/rcmb.2013-0032OC
    1. Trnka J., Blaikie F. H., Logan A., Smith R. A., Murphy M. P. (2009). Antioxidant properties of MitoTEMPOL and its hydroxylamine. Free Radic. Res. 43 4–12. 10.1080/10715760802582183
    1. Tschopp J., Schroder K. (2010). NLRP3 inflammasome activation: the convergence of multiple signalling pathways on ROS production? Nat. Rev. Immunol. 10 210–215. 10.1038/nri2725
    1. Verdia-Baguena C., Nieto-Torres J. L., Alcaraz A., Dediego M. L., Torres J., Aguilella V. M., et al. (2012). Coronavirus E protein forms ion channels with functionally and structurally-involved membrane lipids. Virology 432 485–494. 10.1016/j.virol.2012.07.005
    1. Wang K., Xie S., Sun B. (2011). Viral proteins function as ion channels. Biochim. Biophys. Acta 1808 510–515. 10.1016/j.bbamem.2010.05.006
    1. Wang W., Xiao F., Wan P., Pan P., Zhang Y., Liu F., et al. (2017). EV71 3D protein binds with NLRP3 and enhances the assembly of inflammasome complex. PLoS Pathog. 13:e1006123. 10.1371/journal.ppat.1006123
    1. Wilson L., Mckinlay C., Gage P., Ewart G. (2004). SARS coronavirus E protein forms cation-selective ion channels. Virology 330 322–331. 10.1016/j.virol.2004.09.033
    1. Yu C. J., Chen Y. C., Hsiao C. H., Kuo T. C., Chang S. C., Lu C. Y., et al. (2004). Identification of a novel protein 3a from severe acute respiratory syndrome coronavirus. FEBS Lett. 565 111–116. 10.1016/j.febslet.2004.03.086
    1. Yuan X., Li J., Shan Y., Yang Z., Zhao Z., Chen B., et al. (2005). Subcellular localization and membrane association of SARS-CoV 3a protein. Virus Res. 109 191–202. 10.1016/j.virusres.2005.01.001
    1. Yue Y., Nabar N. R., Shi C. S., Kamenyeva O., Xiao X., Hwang I. Y., et al. (2018). SARS-Coronavirus open reading frame-3a drives multimodal necrotic cell death. Cell Death Dis. 9:904. 10.1038/s41419-018-0917-y
    1. Zeng R., Yang R. F., Shi M. D., Jiang M. R., Xie Y. H., Ruan H. Q., et al. (2004). Characterization of the 3a protein of SARS-associated coronavirus in infected vero E6 cells and SARS patients. J. Mol. Biol. 341 271–279. 10.1016/j.jmb.2004.06.016
    1. Zhou R., Tardivel A., Thorens B., Choi I., Tschopp J. (2010). Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nat. Immunol. 11 136–140. 10.1038/ni.1831
    1. Zhou R., Yazdi A. S., Menu P., Tschopp J. (2011). A role for mitochondria in NLRP3 inflammasome activation. Nature 469 221–225. 10.1038/nature09663

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