猪繁殖与呼吸综合征病毒逃逸Ⅰ型干扰素反应机制的研究

发布时间：2018-04-24 20:40

本文选题：猪繁殖与呼吸综合征病毒 + Ⅰ型干扰素　；参考：《中国农业大学》2016年博士论文

【摘要】：猪繁殖与呼吸综合征是由猪繁殖与呼吸综合征病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)引起的一种高度传染性疾病,给世界养猪业造成了重大经济损失。2006年,我国爆发了高致病性猪繁殖与呼吸综合征,以高热、高发病率和高致死率为典型特征。PRRSV感染能够引起宿主严重的免疫抑制,其中,细胞因子表达紊乱是造成这一现象的主要原因之一。I型干扰素(type I IFNs,IFN-I)是宿主产生的最重要的抗病毒细胞因子,IFN-I不仅可以诱导上百种抗病毒基因的表达直接阻断病毒的复制周期,而且在调节宿主固有免疫和适应性免疫反应中起到非常重要的作用。然而,PRRSV感染却不能很好地诱导IFN-I的表达。为了进一步明确PRRSV致病机理,本论文对高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)逃逸IFN-I的分子机制进行深入的研究。本课题首先用HP-PRRSV感染猪肺泡巨噬细胞(PAMs)和单核细胞趋化的巨噬细胞(BMo),发现HP-PRRSV毒株JXwn06感染可以明显抑制poly(I:C)诱导的IFNβ和IFNa的转录激活,同时也抑制了下游抗病毒基因的表达。应用Western Blot等技术证明HP-PRRSV感染可以抑制NF-κB和IRF3信号通路。筛选HP-PRRSV所有非结构蛋白和结构蛋白后,发现nsp4对poly(I:C)诱导IFNβ的表达有显著的抑制作用。进一步研究发现,nsp4可以抑制IκBα的磷酸化和p65的入核,并下调信号分子RIG-I、VISA、TRIF和IKKβ诱导的NF-κB信号通路的激活,暗示nsp4的作用位点可能在IKK激酶复合体上。通过对IKKα、IKKβ和NEMO (NF-κB信号通路重要调节蛋白)的切割作用发现,nsp4可以特异地靶向NEMO的E349-$350氨基酸对,并对其进行切割,这一作用依赖于nsp4的3C样丝氨酸蛋白酶活性。nsp4的酶活突变体对NEMO和IFNβ的表达均没有影响。同时,我们对HP-PRRSV抑制1RF3的机制也进行了研究,发现nsp4可以切割VISA (RIG样信号通路特异的接头蛋白)使其脱离线粒体膜,从而阻断下游NF-κB和IRF3信号通路,抑制RIG样信号通路介导的抗病毒作用。有趣的是,与传统PRRSV毒株CH-la相比,HP-PRRSV毒株JXwn06的nsp4对poly(I:C)诱导的IFNβ、NF-κB和1RF3的荧光素酶活性的抑制作用更强。进一步研究发现,CH-la感染不能下调VISA的表达,而且CH-la的nsp4对VISA也没有切割作用。这一结果部分揭示了HP-PRRSV毒株与传统毒株致病性存在差异的原因。以上研究结果表明,HP-PRRSV nsp4可以特异性地切割信号通路组分NEMO和VISA,阻断NF-κB和IRF3信号通路,从而抑制IFNβ的转录激活。本论文研究发现了一种新的HP-PRRSV逃逸宿主免疫反应的机制,为进一步阐明HP-PRRSV的致病机理提供了理论基础,具有重要的意义。
[Abstract]:Porcine Reproductive and Respiratory Syndrome virus (PRRS) is a highly contagious disease that has caused significant economic losses to the world pig industry. The outbreak of highly pathogenic porcine reproductive and respiratory syndrome in China is characterized by high fever, high morbidity and high mortality. PRRSv infection can cause severe immunosuppression of the host. The disturbance of cytokine expression is one of the main causes of this phenomenon. Type I interferon type I IFNs IFN-I) is the most important antiviral cytokine produced by the host. IFN-I can not only induce the expression of hundreds of antiviral genes, but also directly block the replication cycle of the virus. Moreover, it plays a very important role in regulating host innate immunity and adaptive immune response. However, PRRSv infection can not induce the expression of IFN-I well. In order to further clarify the pathogenesis of PRRSV, the molecular mechanism of escape IFN-I of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSVV) was studied in this paper. In this study, HP-PRRSV was first used to infect porcine alveolar macrophages (Pam) and monocyte chemotactic macrophages (BMoN). It was found that JXwn06 infection of HP-PRRSV strain could significantly inhibit the transcriptional activation of IFN 尾 and IFNa induced by polymorphic I: C) and also inhibit the expression of downstream antiviral genes. Western Blot and other techniques were used to prove that HP-PRRSV infection could inhibit NF- 魏 B and IRF3 signaling pathway. After screening all the nonstructural proteins and structural proteins of HP-PRRSV, it was found that nsp4 could significantly inhibit the expression of IFN 尾 induced by poly (I: C). It was further found that nsp4 could inhibit the phosphorylation of I 魏 B 伪 and the entry of p65, and down-regulate the activation of NF- 魏 B signaling pathway induced by RIG-IVISATRIF and IKK 尾, suggesting that the site of nsp4 may be on the IKK kinase complex. The cleavage of IKK 伪 -IKK 尾 and NEMO NF- 魏 B signaling pathway showed that nsp4 could specifically target and cleavage the E349-C350 amino acid pairs of NEMO. This effect was dependent on the activity mutants of nsp4 3C like serine protease. Nsp4 had no effect on the expression of NEMO and IFN 尾. At the same time, we also studied the mechanism of inhibition of 1RF3 by HP-PRRSV. It was found that nsp4 could cut the specific junction protein of VISA rig-like signaling pathway and isolate it from mitochondrial membrane, thus blocking the downstream NF- 魏 B and IRF3 signaling pathways. Inhibition of antiviral effects mediated by RIG-like signaling pathway. Interestingly, compared with the traditional PRRSV strain CH-la, the nsp4 of HP-PRRSv strain JXwn06 has a stronger inhibitory effect on the luciferase activity of IFN 尾 -NF- 魏 B and 1RF3. Further studies showed that CH-la infection could not down-regulate the expression of VISA, and nsp4 of CH-la had no effect on VISA cleavage. This result partly revealed the pathogenicity of HP-PRRSV strains and traditional strains. These results suggest that HP-PRRSv nsp4 can specifically cut the signal pathway components NEMO and VISAand block the NF- 魏 B and IRF3 signaling pathways, thus inhibiting the transcriptional activation of IFN 尾. In this paper, we found a new mechanism of immune response to escape host of HP-PRRSV, which provides a theoretical basis for further elucidation of the pathogenesis of HP-PRRSV, which is of great significance.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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