Encephalomyocarditis Virus Viroporin 2B Activates NLRP3 Inflammasome

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 Ca²⁺ level, but not mitochondrial reactive oxygen species and lysosomal cathepsin B, was important in EMCV-induced NLRP3 inflammasome activation. Chelation of extracellular Ca²⁺ did not reduce virus-induced IL-1β secretion. These results indicate that EMCV activates the NLRP3 inflammasome by stimulating Ca²⁺ flux from intracellular storages to the cytosol, and highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. The innate immune system, the first line of defense against invading pathogens, plays a key role not only in limiting microbe replications at early stages of infection, but also in initiating and orchestrating antigen-specific adaptive immune responses. The innate immune responses against viruses usually rely on recognition of viral nucleic acids by host pattern-recognition receptors such as Toll-like receptors and cytosolic helicases. In addition, recent studies have indicated that certain viruses activate the NLRP3 inflammasome, a multiprotein complex containing the intracellular pattern-recognition receptor NLRP3, which in turn induces secretion of proinflammatory cytokines. We have previously revealed the role of the NLRP3 inflammasome in innate recognition of influenza virus, in which the influenza virus proton-selective ion channel M2 protein, but not viral RNA, is required. Here, we demonstrate that another RNA virus, encephalomyocarditis virus (EMCV), also activates the NLRP3 inflammasome in a viral RNAindependent manner. Instead, the EMCV viroporin 2B, which is involved in Ca²⁺ flux from intracellular storages into the cytosol, activates the NLRP3 inflammasome. Our results highlight the importance of viroporins, virusencoded transmembrane pore-forming proteins, in recognition of virus infections by NLRP3.