钙结合蛋白S100A9调节猪繁殖与呼吸综合征病毒引起的炎症反应机制

Porcine reproductive and respiratory syndrome virus (PRRSV) has been an important pathogen threatening the swine industry. PRRSV infection could cause severe interstitial pneumonia. Previous studies have demonstrated that the major agent of interstitial pneumonia is inflammatory responses. However, the mechanism of inflammatory responses is still ill-known. It has been reported that DAMPs play a key role in modulating the inflammatory responses induced by PRRSV. S100A9 protein, a member of calcium binding protein family, is an important molecule of DMAPs, which has been suggested participating in modulating the inflammatory responses induced by some pathogens. We have demonstrated that S100A9 played a role in promoting the inflammatory responses induced by PRRSV using the methods of over-expression or RNA interfere. In the research, we will focus on the mechanism of S100A9 protein in the regulation of inflammatory responses induced by PRRSV. First, we will focus on the role that S100A9 played in the replication of PRRSV using the methods of over-expression or RNA interfere. Then, we will detect the signal pathways associated with inflammatory responses influenced by S100A9. With the method of RNA interfere, we will detect the S100A9 associated PRRs (TLR2, TLR3, TLR4 and RAGE) in PRRSV infection. The effects of S100A9 on the MAPKs, NF-κB signaling pathways will also be detected. Collectively, the research will make clear that the role of S100A9 played in the inflammatory responses and enrich the mechanisms of pathogenesis in PRRSV.

猪繁殖与呼吸综合征病毒（PRRSV）感染可引起猪严重间质性肺炎。钙结合蛋白作为损伤相关分子模式成员能够参与多种病原的炎症损伤，但在PRRSV中尚未见报道。我们前期发现，过表达或siRNA干扰钙结合蛋白S100A9后可明显影响PRRSV引起的炎症反应，表明该蛋白在PRRSV感染中发挥着重要作用。本项目拟通过S100A9蛋白的过表达或干扰，明确其与PRRSV复制的关系。随后，通过siRNA干扰TLR2、TLR3、TLR4与RAGE等模式识别受体（PRRs），检测PRRSV感染后炎症因子的表达，证实在PRRSV感染中与S100A9结合的PRRs分子，并进一步检测相关PRRs的下游接头分子。最后通过S100A9蛋白的过表达或干扰，明确在PRRSV感染中S100A9蛋白对MAPKs和NF-κB通路的影响。本项目将系统阐明S100A9蛋白调节PRRSV引起的炎症反应分子机制，丰富PRRSV的致病机理。

近30年来，猪繁殖与呼吸障碍综合征病毒（PRRSV）一直是世界养猪业的一个大难题。目前，虽然该病毒已有减活疫苗上市，该病毒仍然会有重组、返强的发生，给世界养猪业造成了巨大的损失。因此，进一步了解该病毒与宿主的相互作用，为优化疫苗毒株或清除净化该病毒做理论基础显得尤为重要。近年来，研究表明损伤相关分子机制是病毒致病性的一个重要机制。钙结合蛋白家族蛋白是一类重要的损伤相关分子模式分子。先前研究发现PRRSV感染后会引起钙结合蛋白S100A9蛋白表达的明显升高，表明S100A9可能与PRRSV的复制或致病性相关。本研究进一步发现RRSV感染Marc-145细胞或者猪肺泡巨噬细胞（PAMs）后会引起S100A9在转录水平和蛋白水平的明显升高。同时，我们发现S100A9能够抑制PRRSV的复制，并且S100A9发挥病毒抑制功能是通过钙离子依赖途径的。另外，S100A9能够与PRRSV的核衣壳蛋白（N蛋白）结合，并且共定位在细胞质中；并且在该结合过程中N蛋白的78位和36-37位氨基酸尤为重要。而S100A9的78位氨基酸对PRRSV的复制抑制作用最为关键。最后，我们利用反响遗传技术进一步明确了N蛋白36-37位氨基酸的重要性。因此，该研究表明S100A9能够通过与PRRSV的N蛋白结合从而抑制病毒的复制，进一步阐明PRRSV的相关复制机理，为进一步了解PRRSV致病机制奠定了一定的理论基础。