Encephalomyocarditis virus viroporin 2B activates NLRP3 inflammasome

PLoS Pathog. 2012;8(8):e1002857. doi: 10.1371/journal.ppat.1002857. Epub 2012 Aug 9.

Abstract

Nod-like receptors (NLRs) comprise a large family of intracellular pattern- recognition receptors. Members of the NLR family assemble into large multiprotein complexes, termed the inflammasomes. The NLR family, pyrin domain-containing 3 (NLRP3) is triggered by a diverse set of molecules and signals, and forms the NLRP3 inflammasome. Recent studies have indicated that both DNA and RNA viruses stimulate the NLRP3 inflammasome, leading to the secretion of interleukin 1 beta (IL-1β) and IL-18 following the activation of caspase-1. We previously demonstrated that the proton-selective ion channel M2 protein of influenza virus activates the NLRP3 inflammasome. However, the precise mechanism by which NLRP3 recognizes viral infections remains to be defined. Here, we demonstrate that encephalomyocarditis virus (EMCV), a positive strand RNA virus of the family Picornaviridae, activates the NLRP3 inflammasome in mouse dendritic cells and macrophages. Although transfection with RNA from EMCV virions or EMCV-infected cells induced robust expression of type I interferons in macrophages, it failed to stimulate secretion of IL-1β. Instead, the EMCV viroporin 2B was sufficient to cause inflammasome activation in lipopolysaccharide-primed macrophages. While cells untransfected or transfected with the gene encoding the EMCV non-structural protein 2A or 2C expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells transfected with the gene encoding the EMCV 2B or influenza virus M2 protein. 2B proteins of other picornaviruses, poliovirus and enterovirus 71, also caused the NLRP3 redistribution. Elevation of the intracellular Ca(2+) level, but not mitochondrial reactive oxygen species and lysosomal cathepsin B, was important in EMCV-induced NLRP3 inflammasome activation. Chelation of extracellular Ca(2+) did not reduce virus-induced IL-1β secretion. These results indicate that EMCV activates the NLRP3 inflammasome by stimulating Ca(2+) flux from intracellular storages to the cytosol, and highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / metabolism*
  • Cardiovirus Infections / genetics
  • Cardiovirus Infections / metabolism*
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Encephalomyocarditis virus / genetics
  • Encephalomyocarditis virus / metabolism*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Inflammasomes / genetics
  • Inflammasomes / metabolism*
  • Interleukin-18 / genetics
  • Interleukin-18 / metabolism
  • Interleukin-1beta / genetics
  • Interleukin-1beta / metabolism
  • Macrophages / metabolism
  • Macrophages / virology
  • Mice
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • RNA, Viral / genetics
  • RNA, Viral / metabolism
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*
  • Virion / genetics
  • Virion / metabolism

Substances

  • Carrier Proteins
  • Inflammasomes
  • Interleukin-18
  • Interleukin-1beta
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • Nlrp3 protein, mouse
  • RNA, Viral
  • Viral Proteins
  • Calcium

Grant support

This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology, and the Ministry of Health, Labor, and Welfare of Japan; the Kanae Foundation for the Promotion of Medical Science; and Takeda Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.