PRRSV云南分离株抑制I型干扰素产生的RIG-I/MDA5信号转导通路研究

Recently there has been an increase in research focused on the cell signal pathway in viral disease pathogenesis, which is a novel and major trend. However, The role of RIG-I/MDA5 signal pathway during the inhibition of type I IFN by PRRSV remains unclear. In this study, PAMs infected with PRRSV Yunnan local strain (YN-2011) will be collected at different time points post infection as the experiment material. This study will explore the relations between PRRSV mRNA transcription and the type I IFN production through the RIG-I/MDA5 pathway. Analysis of the RIG-I/MDA5 pathway will include evaluation of the dynamic mRNA transcription, the protein expression or phosphorylation of individual signal genes, and the differences in transmembrane protein translocation and distribution between nuclear or mitochondrial membrane and the cytoplasm. These variables will be explored by the ELISA, Real-time PCR, Western Blot and Laser scanning confocal microscope technology. Furthermore, the effects of the crucial signal genes will be verified by transfecting cells. We expect to elucidate the molecular mechanism of PRRSV type I IFN inhibition within the cell signal pathway, which will potentially provide the theoretical and technological support for revealing the pathogenesis of innate immune suppression. These results will provide very important scientific knowledge to enrich and improve our understanding of the interaction between PRRSV and host cells.

细胞信号转导通路在病毒性疾病中的致病机理日益受到重视，现已成为一个新的研究方向和热点，但目前有关PRRSV抑制PAM细胞产生I型IFN的RIG-I/MDA5信号转导通路文献在国内外鲜有报道。本研究拟用ELISA、Real-time PCR、Western Blot、激光共聚焦显微镜等技术，探讨PRRSV云南分离株YN-2011感染PAM后不同时间段病毒基因转录水平与I型IFN产量的关系、RIG-I/MDA5信号通路中各种受体基因mRNA转录情况、信号蛋白表达与磷酸化的动态变化、跨膜蛋白在细胞核膜或线粒体膜定位分布改变对I型IFN的影响，并通过细胞转染信号基因反证其调节作用。以期从信号转导通路的角度阐明PRRSV抑制I型IFN产生的分子机制，为揭示PRRSV感染导致先天性免疫抑制的致病机理提供理论依据和技术支撑，这对于丰富并提升PRRSV与宿主细胞相互作用的认识水平具有重要的科学意义。

为探讨PRRSV体外感染猪肺泡巨噬细胞（PAMs）后对I型干扰素及RIG-I/MDA5信号通路的影响，研究选取4周龄PRRSV阴性且抗体阴性的健康仔猪，用于采集PAMs。将收集的细胞分为空白对照组、Poly(I:C)处理组、PRRSV感染组、Poly(I:C)+PRRSV组和PRRSV+Poly(I:C)组，分别于不同时间点（0、6、12、24、36、48和60h）收集各组细胞及细胞培养上清液，用MTS一步法检测各组细胞活力；用酶联免疫法（ELISA）检测细胞培养上清中干扰素α（IFN-α）、干扰素β（IFN-β）的表达变化；应用实时荧光定量PCR法检测IFN-α、IFN-β、干扰素调节因子3（Interferon regulatory factor 3, IRF3）、干扰素调节因子7（Interferon regulatory factor 7, IRF7）、维甲酸诱导基因I（RIG-I）、黑素瘤差异化相关基因5（MDA5）、线粒体抗病毒信号分子（MAVS）、PRRSV核衣壳蛋白基因（N蛋白基因）、β-actin的mRNA转录水平变化。结果显示，（1）PRRSV感染组以及PRRSV+Poly(I:C)组细胞活力较正常组的明显下降(P<0.01)。（2）PRRSV感染组以及PRRSV+Poly(I:C)组细胞培养上清中的IFN-β生成量与正常对照组相比显著减少(P<0.01)，而Poly(I:C)处理组以及Poly(I:C)+ PRRSV组细胞上清中的IFN-β和IFN-α生成量增加。（3）在基因转录水平，PRRSV感染组以及PRRSV+Poly(I:C)组中的IFN-β、IRF3、RIG-I和MAVS的mRNA转录水平显著下调(P<0.01)，PRRSV mRNA转录水平显著上调(P<0.01)；在Poly(I:C)处理组以及Poly(I:C)+ PRRSV组中，IFN-β、IRF、RIG-I、IFN-α以及IRF7的mRNA转录水平上调，但Poly(I:C)+ PRRSV组中的PRRSV mRNA转录水平较PRRSV感染组明显下调(P<0.01)。以上结果表明，PRRSV能够导致PAMs活力降低；PRRSV能够抑制PAMs分泌IFN-β，从而使PAMs先天性免疫功能受到抑制，其机制是PRRSV下调IFN-β、IRF3、RIG-I、MAVS基因的转录水平而实现的。