真核生物翻译起始因子5A调控PRRSV复制的分子机制研究

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). After infecting host cells, PRRSV recruits host translation elements to synthesize its own proteins. My previous research confirmed that eukaryotic translation initiation factor 5A (eIF5A) and other translation -related proteins were significantly down-regulated in porcine alveolar macrophages (PAMs) after PRRSV infection. Knockdown expression of eIF5A using RNAi technology in vitro can significantly inhibit PRRSV replication, which suggests that eIF5A may play a key role in regulating PRRSV replication. The purpose of this study is to analyze the effect of eIF5A hypusination on PRRSV replication using hypusinated specific inhibitors.By co-immunoprecipitation and mass spectrometry techniques, the viral proteins interacting with eIF5A and their key amino acid sequences were analyzed. The role of mTOR signal pathway in mediating regulation of eIF5A in PRRSV replication was further clarified through the use of small molecule inhibitors. This study will give insight to the molecular mechanism of eIF5A regulating PRRSV replication, and will provide a theoretical basis for the prevention and control of PRRSV.

猪繁殖与呼吸综合征（PRRS）是由猪繁殖与呼吸综合征病毒（PRRSV）引起的高度接触性传染病。PRRSV感染宿主细胞后必须征用宿主翻译元件合成自身蛋白。申请人前期研究证实PRRSV感染猪肺泡巨噬细胞（PAMs）后，真核生物翻译起始因子5A（eIF5A）等翻译相关蛋白显著下调表达。采用RNAi技术体外敲低eIF5A能显著抑制PRRSV的复制，推测eIF5A在调控PRRSV复制过程中发挥关键作用。项目拟使用羟脯氨酸修饰特异性抑制剂，分析eIF5A羟脯氨酸修饰对PRRSV复制的影响；免疫共沉淀及质谱技术解析与eIF5A互作的病毒蛋白及其关键氨基酸序列，使用小分子抑制剂进一步明确mTOR信号通路在介导eIF5A调控PRRSV复制中的作用。研究将阐明eIF5A调控PRRSV复制的分子机制，为PRRSV的防控提供理论依据。

猪繁殖与呼吸综合征（PRRS）是由猪繁殖与呼吸综合征病毒（PRRSV）引起的高度接触性传染病，在世界范围内给养猪业造成巨大经济损失。为明确真核翻译起始因子5A（eIF5A）及其羟脯氨酸修饰在调控PRRSV复制中的作用以及阐明mTOR信号通路在介导eIF5A调控PRRSV复制过程中的信号转导机制。本研究通过慢病毒载体系统构建稳定表达eIF5A的MARC-145-eIF5A细胞系，使用CRISPR/Cas9技术构建敲除eIF5A的MARC-145-△eIF5A细胞系。通过IFA、Real-time PCR、Western blot和TCID50试验证实体外稳定表达eIF5A显著促进PRRSV的增殖，而敲除eIF5A显著抑制PRRSV增殖。eIF5A是唯一含有羟脯氨酸修饰的蛋白，羟脯氨酸修饰对于eIF5A 的功能至关重要。使用eIF5A羟脯氨酸修饰特异性小分子抑制剂CNI-1493，GC7，Ciclopirox处理细胞后感染PRRSV，通过IFA、Real-time PCR、Western blot和TCID50试验证实三种小分子抑制剂均可抑制PRRSV的增殖。eIF5A是mTOR信号通路下游的信号分子，在PRRSV感染PAMs后用Western Blot检测mTOR信号通路下游信号分子的表达及磷酸化情况，结果表明PRRSV感染PAMs激活mTOR信号通路，mTOR小分子抑制剂Rapamycin能消除PRRSV感染引起的4EBP1磷酸化和eIF5A表达抑制，促进PRRSV的增殖，说明mTOR信号通路在介导eIF5A调控PRRSV复制过程中发挥重要作用。综上所述，本研究为深入理解PRRSV的致病机制奠定理论基础，同时也为抗PRRSV新型药物筛选提供新的思路和借鉴。