Encephalomyocarditis Virus Viroporin 2B Activates NLRP3 Inflammasome

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.

核苷酸结合寡聚化结构域样受体（Nod-like receptors, NLRs）是一类庞大的细胞内模式识别受体家族。NLR家族成员可组装形成大型多蛋白复合体，即炎性体（inflammasomes）。含 pyrin 结构域的 NLR 家族3（NLRP3）可被多种分子与信号触发，进而形成 NLRP3 炎性体。近期研究表明，DNA病毒与RNA病毒均可激活 NLRP3 炎性体，经半胱天冬氨酸蛋白酶1（caspase-1）活化后，介导白细胞介素1β（IL-1β）与白细胞介素18（IL-18）的分泌。本团队前期研究证实，流感病毒的质子选择性离子通道M2蛋白可激活 NLRP3 炎性体。然而，NLRP3识别病毒感染的确切机制仍有待阐明。本研究证实，脑心肌炎病毒（encephalomyocarditis virus, EMCV）——一种隶属于小RNA病毒科（Picornaviridae）的正链RNA病毒——可在小鼠树突状细胞与巨噬细胞中激活 NLRP3 炎性体。尽管转染脑心肌炎病毒粒子或脑心肌炎病毒感染细胞的RNA，可在巨噬细胞中有效诱导I型干扰素的表达，但无法刺激IL-1β的分泌。与之相反，脑心肌炎病毒的病毒孔蛋白2B足以在脂多糖预激活的巨噬细胞中诱发炎性体活化。未转染或转染脑心肌炎病毒非结构蛋白2A或2C编码基因的细胞，其NLRP3在胞质内均匀分布；而转染脑心肌炎病毒2B或流感病毒M2蛋白编码基因的细胞中，NLRP3会重新分布至核周间隙。其他小RNA病毒（如脊髓灰质炎病毒与肠道病毒71型）的2B蛋白，同样可引发NLRP3的重新分布。在脑心肌炎病毒诱导的NLRP3炎性体活化过程中，细胞内钙离子水平升高发挥了关键作用，而线粒体活性氧与溶酶体组织蛋白酶B则无此必要性。使用细胞外钙离子螯合剂并未降低病毒诱导的IL-1β分泌水平。上述结果表明，脑心肌炎病毒通过促进细胞内储存的钙离子向胞质流动，激活 NLRP3 炎性体；本研究同时凸显了病毒孔蛋白——一类跨膜成孔病毒蛋白——在病毒诱导的NLRP3炎性体活化中的重要性。