DDX19A识别PRRSV基因组RNA并激活NLRP3炎症小体
发布时间：2018-09-12 12:29
【摘要】：猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(PRRS virus,PRRSV)引起的一类严重接触性传染病,主要引起怀孕母猪流产、早产、死胎、木乃伊胎及仔猪呼吸道疾病等症状。2006年爆发的由高致病PRRSV(highly pathogenic PRRSV,HP-PRRSV)引起的以高热、高发病率和高死亡率为特征的高致病PRRS给我国的生猪产业造成了难以估量的经济损失。HP-PRRSV感染猪肺泡巨噬细胞(pulmonary alveolar macrophages,PAMs),诱导促炎性细胞因子的大量分泌,引起严重的肺部炎症症状。我们实验室前期研究结果证明弱毒株CH1a、CH1R和疫苗毒株Hu N4-F112均不能诱导IL-1β和TNF-α的分泌,而HP-PRRSV毒株Hu N4和SDA2却均能诱导这些细胞因子的高水平表达和分泌,表明强弱毒株诱导炎症反应的能力不同。IL-1β作为最重要的炎症介质之一,在启动和放大炎症反应,清除感染,加速组织修复,活化保护性免疫反应等过程中发挥重要作用。大量研究表明,HP-PRRSV感染可以诱导IL-1β的分泌,但是,具体的分子机制还不清楚。本研究从分子水平探究HP-PRRSV感染诱导IL-1β分泌的机理。IL-1β的成熟需要两个信号:第一信号由细胞编码的模式识别受体(Pattern recognition receptor,PRRs)启动NF-κB信号通路活化,促进pro-IL-1β的产生;第二信号由病原相关分子模式(Pathogen-associated molecular patterns,PAMPs)或危险相关分子模式(Damage Associated Molecular Patterns,DAMPs)介导炎症小体激活,引起pro-caspase-1活化及pro-IL-1β成熟分泌。本研究证明,HP-PRRSV感染可以诱导pro-caspase-1的活化以及pro-IL-1β的切割成熟。深入研究发现HP-PRRSV感染可以诱导NLRP3炎症小体复合物的形成。敲低NLRP3的表达显著抑制PRRSV感染诱导的pro-caspase-1活化和IL-1β分泌。随后的研究发现HP-PRRSV基因组RNA可以诱导较高水平的IL-1β分泌,证明HP-PRRSV基因组RNA及其5?UTR转录物均可以诱导NLRP3炎症小体的激活。敲低NLRP3的表达显著抑制HP-PRRSV基因组RNA及其5?UTR转录物诱导的IL-1β分泌。本研究用大量数据证明了HP-PRRSV感染可以激活NLRP3炎症小体,且发现HP-PRRSV基因组RNA就足以激活NLRP3炎症小体,其诱导IL-1β分泌与病毒的复制过程无关。病毒RNA可以诱导NLRP3炎症小体的活化,但是目前还没有任何证据证明NLRP3炎症小体的组分可以直接识别病毒的RNA。因此,我们推测细胞内可能存在未知的病毒RNA受体分子。利用HP-PRRSV感染猪PAMs为模型,通过抗ASC抗体免疫共沉淀试验和串联质谱分析鉴定出了DDX19A。我们发现DDX19A与NLRP3炎症小体复合物中的NLRP3相互作用,而不与ASC和caspase-1相互作用。进一步证明DDX19A能直接结合HP-PRRSV基因组RNA及其5?UTR和3?UTR转录物、poly I:C。敲低DDX19A的表达显著抑制HP-PRRSV基因组RNA及其5?UTR转录物诱导的NLRP3炎症小体活化和IL-1β分泌,但不影响poly I:C和LPS/ATP诱导的IL-1β分泌。综上所述,本研究从分子水平揭示了HP-PRRSV感染PAMs诱导NLRP3炎症小体激活的机理,证明宿主蛋白DDX19A可以识别HP-PRRSV基因组RNA,招募NLRP3,激活NLRP3炎症小体,诱导IL-1β分泌。本研究的创新性结果为防控PRRS和开发抗炎药物提供重要参考。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRS) is a serious * * contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRS virus), which mainly causes pregnancy, sow abortion, premature birth, stillbirth, mummification * and piglet respiratory diseases. V (highly pathogenic PRRSV, HP-PRRSV), a highly pathogenic PRRS characterized by high fever, high morbidity and high mortality, has caused incalculable economic losses to the pig industry in China..HP-PRRSV infection of * * porcine alveolar macrophages (pulmonary alveolar macrophages, PAMs) has induced severe secretion of proinflammatory cytokines, causing severe morbidity. Previous studies in our laboratory showed that attenuated strains CH1a, CH1R and vaccine strains Hu N4-F112 could not induce the secretion of IL-1beta and TNF-alpha, while HP-PRRSV strains Hu N4 and SDA2 could induce the high level expression and secretion of these cytokines, indicating that the ability of strong and weak strains to induce inflammation was different. One of the important inflammatory mediators plays an important role in initiating and amplifying inflammatory response, clearing infection, accelerating tissue repair and activating protective immune response. A large number of studies have shown that HP-PRRSV infection can induce IL-1 beta secretion, but the specific molecular mechanism is still unclear. Mechanisms of IL-1 beta secretion. The maturation of IL-1 beta requires two signals: the first signal is activated by cell-encoded pattern recognition receptors (PRRs) to activate the NF-kappa B signaling pathway and promote pro-IL-1 beta production; the second signal is either Pathogen-associated molecular patterns (PAMPs) or risk-related. Damage Associated Molecular Patterns (DAMPs) mediate the activation of inflammatory corpuscles, induce the activation of pro-caspase-1 and the maturation and secretion of pro-IL-1 beta. This study demonstrates that HP-PRRSV infection can induce the activation of pro-caspase-1 and the cleavage and maturation of pro-IL-1 beta. Inhibition of NLRP3 expression significantly inhibited pro-caspase-1 activation and IL-1 beta secretion induced by PRRSV infection. Subsequent studies found that HP-PRRSV genomic RNA could induce a higher level of IL-1 beta secretion, suggesting that both HP-PRRSV genomic RNA and its 5?UTR transcripts could induce the activation of NLRP3 inflammatory bodies. P-PRRSV genomic RNA and its 5?UTR transcripts induce IL-1 beta secretion. This study demonstrated that HP-PRRSV infection can activate NLRP3 inflammation bodies, and found that HP-PRRSV genomic RNA is sufficient to activate NLRP3 inflammation bodies, and its induction of IL-1 beta secretion has nothing to do with the replication process of the virus. Viral RNA can induce the activation of NLRP3 inflammation bodies. However, there is no evidence that the components of NLRP3 inflammable bodies can directly recognize the RNA. of the virus. Therefore, we speculate that there may be unknown RNA receptor molecules in the cell. * using HP-PRRSV infected pig PAMs as a model, we identified DDX19A. by anti ASC antibody immunoprecipitation and tandem mass spectrometry, and we found DDX. It is further demonstrated that DDX19A can bind directly to HP-PRRSV genomic RNA and its 5?UTR and 3?UTR transcripts, and poly I:C. knockdown of DDX19A significantly inhibits the activation of NLRP3 inflammatory bodies induced by HP-PRRSV genomic RNA and its 5?UTR transcripts and IL-1. In conclusion, this study revealed the mechanism of activation of NLRP3 inflammatory bodies induced by HP-PRRSV infection with PAMs at the molecular level, and demonstrated that host protein DDX19A could recognize HP-PRRSV genomic RNA, recruit NLRP3, activate NLRP3 inflammatory bodies and induce IL-1 beta secretion. The results provide important reference for preventing and controlling PRRS and developing anti-inflammatory drugs.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28
本文编号：2239010
[Abstract]:Porcine reproductive and respiratory syndrome (PRRS) is a serious * * contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRS virus), which mainly causes pregnancy, sow abortion, premature birth, stillbirth, mummification * and piglet respiratory diseases. V (highly pathogenic PRRSV, HP-PRRSV), a highly pathogenic PRRS characterized by high fever, high morbidity and high mortality, has caused incalculable economic losses to the pig industry in China..HP-PRRSV infection of * * porcine alveolar macrophages (pulmonary alveolar macrophages, PAMs) has induced severe secretion of proinflammatory cytokines, causing severe morbidity. Previous studies in our laboratory showed that attenuated strains CH1a, CH1R and vaccine strains Hu N4-F112 could not induce the secretion of IL-1beta and TNF-alpha, while HP-PRRSV strains Hu N4 and SDA2 could induce the high level expression and secretion of these cytokines, indicating that the ability of strong and weak strains to induce inflammation was different. One of the important inflammatory mediators plays an important role in initiating and amplifying inflammatory response, clearing infection, accelerating tissue repair and activating protective immune response. A large number of studies have shown that HP-PRRSV infection can induce IL-1 beta secretion, but the specific molecular mechanism is still unclear. Mechanisms of IL-1 beta secretion. The maturation of IL-1 beta requires two signals: the first signal is activated by cell-encoded pattern recognition receptors (PRRs) to activate the NF-kappa B signaling pathway and promote pro-IL-1 beta production; the second signal is either Pathogen-associated molecular patterns (PAMPs) or risk-related. Damage Associated Molecular Patterns (DAMPs) mediate the activation of inflammatory corpuscles, induce the activation of pro-caspase-1 and the maturation and secretion of pro-IL-1 beta. This study demonstrates that HP-PRRSV infection can induce the activation of pro-caspase-1 and the cleavage and maturation of pro-IL-1 beta. Inhibition of NLRP3 expression significantly inhibited pro-caspase-1 activation and IL-1 beta secretion induced by PRRSV infection. Subsequent studies found that HP-PRRSV genomic RNA could induce a higher level of IL-1 beta secretion, suggesting that both HP-PRRSV genomic RNA and its 5?UTR transcripts could induce the activation of NLRP3 inflammatory bodies. P-PRRSV genomic RNA and its 5?UTR transcripts induce IL-1 beta secretion. This study demonstrated that HP-PRRSV infection can activate NLRP3 inflammation bodies, and found that HP-PRRSV genomic RNA is sufficient to activate NLRP3 inflammation bodies, and its induction of IL-1 beta secretion has nothing to do with the replication process of the virus. Viral RNA can induce the activation of NLRP3 inflammation bodies. However, there is no evidence that the components of NLRP3 inflammable bodies can directly recognize the RNA. of the virus. Therefore, we speculate that there may be unknown RNA receptor molecules in the cell. * using HP-PRRSV infected pig PAMs as a model, we identified DDX19A. by anti ASC antibody immunoprecipitation and tandem mass spectrometry, and we found DDX. It is further demonstrated that DDX19A can bind directly to HP-PRRSV genomic RNA and its 5?UTR and 3?UTR transcripts, and poly I:C. knockdown of DDX19A significantly inhibits the activation of NLRP3 inflammatory bodies induced by HP-PRRSV genomic RNA and its 5?UTR transcripts and IL-1. In conclusion, this study revealed the mechanism of activation of NLRP3 inflammatory bodies induced by HP-PRRSV infection with PAMs at the molecular level, and demonstrated that host protein DDX19A could recognize HP-PRRSV genomic RNA, recruit NLRP3, activate NLRP3 inflammatory bodies and induce IL-1 beta secretion. The results provide important reference for preventing and controlling PRRS and developing anti-inflammatory drugs.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28
【参考文献】
相关硕士学位论文 前1条
1 刘园园;猪繁殖与呼吸综合征病毒激活NLRP3炎症小体的研究[D];西北农林科技大学;2014年
，本文编号：2239010
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2239010.html
