Wen Xuexia, Bian Ting, Zhang Zhibang, Zhou Lei, Ge Xinna, Han Jun, Guo Xin, Yang Hanchun, Yu Kangzhen
Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agrobiotechnology, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District, Beijing, 100193, People's Republic of China.
The Chinese Ministry of Agriculture, No.11 Nongzhanguan Nanli, Chaoyang District, Beijing, 100125, People's Republic of China.
Virol J. 2017 Jul 11;14(1):125. doi: 10.1186/s12985-017-0794-5.
Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failures in sows and respiratory diseases in growing pigs, resulting in huge economic loss for the pig production worldwide. The nonstructural protein 9 (nsp9) and nonstructural protein 2 (nsp2) of PRRSV are known to play important roles in viral replication. Cellular interleukin-2 enhancer binding factor 2 (ILF2) participates in many cellular pathways and involves in life cycle of some viruses. In the present study, we analyzed the interaction of cellular ILF2 with the nsp9 and nsp2 of PRRSV in vitro and explored the effect of ILF2 on viral replication.
The interaction of ILF2 with the nsp9 or nsp2 of PRRSV was analyzed in 293FT cells and MARC-145 cells by co-immunoprecipitation (Co-IP) and the co-localization of ILF2 with the nsp9 or nsp2 of PRRSV in MARC-145 cell and pulmonary alveolar macrophages (PAMs) was examined by confocal immunofluorescence assay. The effect of ILF2 knockdown and over-expression on PRRSV replication was explored in MARC-145 cells by small interfering RNA (siRNA) and lentivirus transduction, respectively.
The interaction of ILF2 with nsp9 or nsp2 was first demonstrated in 293FT cells co-transfected with ILF2-expressing plasmid and nsp9-expressing plasmid or nsp2-expressing plasmid. The interaction of endogenous ILF2 with the nsp9 or nsp2 of PRRSV was further confirmed in MARC-145 cells transduced with GFP-nsp9-expressing lentiviruses or infected with PRRSV JXwn06. The RdRp domain of nsp9 was shown to be responsible for its interaction with ILF2, while three truncated nsp2 were shown to interact with ILF2. Moreover, we observed that ILF2 partly translocated from the nucleus to the cytoplasm and co-localized with nsp9 and nsp2 in PRRSV-infected MARC-145 cells and PAMs. Finally, our analysis indicated that knockdown of ILF2 favored the replication of PRRSV, while over-expression of ILF2 impaired the viral replication in MARC-145 cells.
Our findings are the first to confirm that the porcine ILF2 interacts with the nsp9 and nsp2 of PRRSV in vitro, and exerts negatively regulatory effect on the replication of PRRSV. Our present study provides more evidence for understanding the roles of the interactions between cellular proteins and viral proteins in the replication of PRRSV.
猪繁殖与呼吸综合征病毒（PRRSV）可导致母猪繁殖失败及生长猪呼吸道疾病，给全球养猪业造成巨大经济损失。已知PRRSV的非结构蛋白9（nsp9）和非结构蛋白2（nsp2）在病毒复制中起重要作用。细胞白细胞介素2增强子结合因子2（ILF2）参与许多细胞途径，并涉及某些病毒的生命周期。在本研究中，我们分析了细胞ILF2与PRRSV的nsp9和nsp2在体外的相互作用，并探讨了ILF2对病毒复制的影响。
通过免疫共沉淀（Co-IP）在293FT细胞和MARC-145细胞中分析ILF2与PRRSV的nsp9或nsp2的相互作用，并通过共聚焦免疫荧光测定法检测ILF2与PRRSV的nsp9或nsp2在MARC-145细胞和肺泡巨噬细胞（PAM）中的共定位。分别通过小干扰RNA（siRNA）和慢病毒转导在MARC-145细胞中探讨ILF2敲低和过表达对PRRSV复制的影响。
首次在共转染表达ILF2的质粒和表达nsp9的质粒或表达nsp2的质粒的293FT细胞中证实了ILF2与nsp9或nsp2的相互作用。在用表达GFP-nsp9的慢病毒转导或感染PRRSV JXwn06的MARC-145细胞中进一步证实了内源性ILF2与PRRSV的nsp9或nsp2的相互作用。nsp9的RdRp结构域被证明负责其与ILF2的相互作用，而三个截短的nsp2被证明与ILF2相互作用。此外，我们观察到在PRRSV感染的MARC-145细胞和PAM中，ILF2部分从细胞核转移到细胞质并与nsp9和nsp2共定位。最后，我们的分析表明，敲低ILF2有利于PRRSV的复制，而ILF2的过表达则损害了MARC-145细胞中的病毒复制。
我们的研究结果首次证实猪ILF2在体外与PRRSV的nsp9和nsp2相互作用，并对PRRSV的复制发挥负调控作用。我们目前的研究为理解细胞蛋白与病毒蛋白之间的相互作用在PRRSV复制中的作用提供了更多证据。
