DDX19A Senses PRRSV Genomic RNA And Activates NLRP3 Inflammasome

Porcine reproductive and respiratory syndrome(PRRS), which characterized by the abortion, premature birth, stillbirth and fetal mummies of pregnant sows and the respiratory diseases of piglets, is a serious contagious disease caused by PRRS virus(PRRSV). In 2006, the outbreak of the highly pathogenic PRRS which was caused by the highly pathogenic PRRSV(HP-PRRSV) and characterized by high fever, high morbidity and high mortality in China disastrously attacked our country’s swine industry and spread to the adjacent countries, which have caused uncountable losses. HP-PRRSV infection induces severe inflammation in the lungs, high levels of proinflammatory cytokines can be detected in the early stage of the infection. Previous studies showed that piglets infected with different PRRSV strains produced different levels and types of cytokines, such as IFN-γ, IL-1β, IL-6 and TNF-α. We infected porcine pulmonary alveolar macrophages(PAMs) with different PRRSV strains and found that the infection of the lower virulent PRRSV strains such as CH1 a and its vaccine strain CH1 R and Hu N4-F112, a virulence-attenuated strain of Hu N4, barely induced the m RNA expression and secretion of IL-1β and TNF-α. However, the infection of the HP-PRRSV strains such as Hu N4 and SDA2 stimulated robust expression and secretion of these two cytokines. IL-1β, one of the most important mediators of inflammation, plays critical roles in initiating and magnifying inflammatory responses, antagonizing infections, promoting tissue repairs and activating protective immune responses. Previous studies showed that HP-PRRSV infection induced IL-1β secretion,but the underlying mechanism remains unknown. The aim of the study is to decipher the mechanism of HP-PRRSV infection-induced IL-1β secretion in PAMs.The maturation of pro-IL-1β requires two signals: the signaling 1, which is initiated by the pattern recognition receptors(PRRs), leads to the activation of NF-κB signaling pathway and production of the pro-IL-1β; the signaling 2, which is triggered by the pattern-associated molecular patterns(PAMPs) or the danger-associated molecular patterns(DAMPs), leading to the activation of inflammasome, cleavage of pro-caspase-1, maturation and secretion of pro-IL-1β. In this study, we demonstrated that HP-PRRSV infection induced the cleavage of pro-caspase-1 and maturation of pro-IL-1β. Then, we revealed that HP-PRRSV infection induced the formation of NLRP3 inflammasome complex and knockdown of NLRP3 expression significantly inhibited the HP-PRRSV infection-induced pro-caspase-1 cleavage and IL-1β secretion. Further, we found that HP-PRRSV Hu N4 genomic RNA could induce the secretion of IL-1β. We proved that both HP-PRRSV genomic RNA and its 5?UTR transcripts could trigger the formation of NLRP3 inflammasome complex, cleavage of pro-caspase-1 and maturation of pro-IL-1β. Knockdown of NLRP3 expression significantly inhibited HP-PRRSV genomic RNA- and its 5?UTR transcripts-induced IL-1β secretion. Taken together, we demonstrated that HP-PRRSV infection could activate NLRP3 inflammasome and HP-PRRSV genomic RNA could sufficiently induce NLRP3-dependent IL-1β secretion which is independent of HP-PRRSV replication in vitro.It has been reported that viral RNA could induce the activation of NLRP3 inflammasome. However, there is no data so far to prove that any component of NLRP3 inflammasome can directly sense viral RNAs. Thus, we speculated that there might exist unknown RNA sensors to mediate NLRP3 inflammasome activation. In this study, using HP-PRRSV infection as the model, we identified DDX19 A as a novel RNA sensor through co-immunoprecipitation(Co-IP) and tandem mass spectrometry(MS). We found that DDX19 A exclusively interacted with NLRP3, but not ASC or caspase-1. Further, we proved that DDX19 A could directly bind HP-PRRSV Hu N4 genomic RNA and its 5?UTR and 3?UTR transcripts, poly I:C, bacterial RNA. Knockdown of DDX19 A expression significantly inhibited HP-PRRSV genomic RNA- and its 5?UTR transcripts-induced NLRP3 inflammasome activation and IL-1β secretion.In this study, we decipered the mechanism of HP-PRRSV infection-induced NLRP3 inflammasome activation from the molecular level, proved that DDX19 A could sense HP-PRRSV genomic RNA, recruit NLRP3 and activate NLRP3 inflammasome, thus leading to IL-1β secretion. These innovative results provide important references for PRRS prevention and anti-inflammatory drugs development.