Wang Haifei, Bi Zhenbin, Dai Kaiyu, Li Pinghua, Huang Ruihua, Wu Shenglong, Bao Wenbin
Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design, College of Animal Science and Technology, Yangzhou University, Yangzhou, China.
Institute of Swine Science, Nanjing Agricultural University, Nanjing, China.
Front Microbiol. 2022 Jun 13;13:877644. doi: 10.3389/fmicb.2022.877644. eCollection 2022.
Porcine epidemic diarrhea virus (PEDV) causes a highly contagious intestinal disease in neonatal pigs. Aquaporin-3 (AQP3) plays important roles in maintenance of intestinal barrier function and regulation of immune responses. However, the roles of AQP3 in mediating PEDV infection to host cells and the regulatory mechanisms of AQP3 expression remain poorly understood. Here, we identified one 16 bp (GGGCGGGGTTGCGGGC) insertion mutation in the AQP3 gene promoter in Large White pigs, with the frequencies of 49.3% of heterozygotes and 31.3% of mutant homozygotes. Functional analysis by luciferase activity assay indicated that the insertion mutation results in significant enhancement in AQP3 transcriptional activity ( < 0.01). Mechanistic analysis showed that the inserted sequence adds binding sites for transcription factor CEBPA, which promotes the expression of AQP3. Downregulation of AQP3 by shRNA silencing in porcine intestinal epithelial cells revealed obvious increases in genome copies and viral titers of PEDV. Expression of proinflammatory cytokines (IL-6, IL-8, and IL-18) and interferons (IFN-α and IFN-β) were significantly reduced ( < 0.01) in AQP3 knockdown cells upon PEDV infection. Furthermore, decreased level of ZO-1 protein was also detected in AQP3 knockdown cells in response to PEDV infection. Our findings suggested a previously unknown mechanism linking the effects of promoter genetic variants on the expression of AQP3, revealed the roles of AQP3 in response to PEDV pathogenesis, and indicated the potential associations of the 16 bp insertion mutation with resistance to PEDV infection in porcine intestinal epithelial cells.
猪流行性腹泻病毒（PEDV）可在新生仔猪中引发一种高度传染性的肠道疾病。水通道蛋白3（AQP3）在维持肠道屏障功能和调节免疫反应中发挥着重要作用。然而，AQP3在介导PEDV感染宿主细胞中的作用以及AQP3表达的调控机制仍知之甚少。在此，我们在大白猪的AQP3基因启动子中鉴定出一个16 bp（GGGCGGGGTTGCGGGC）的插入突变，杂合子频率为49.3%，突变纯合子频率为31.3%。荧光素酶活性测定的功能分析表明，该插入突变导致AQP3转录活性显著增强（<0.01）。机制分析显示，插入序列增加了转录因子CEBPA的结合位点，从而促进了AQP3的表达。通过shRNA沉默猪肠道上皮细胞中的AQP3，发现PEDV的基因组拷贝数和病毒滴度明显增加。在PEDV感染后，AQP3敲低细胞中促炎细胞因子（IL-6、IL-8和IL-18）和干扰素（IFN-α和IFN-β）的表达显著降低（<0.01）。此外，在PEDV感染的AQP3敲低细胞中还检测到紧密连接蛋白1（ZO-1）蛋白水平降低。我们的研究结果揭示了一种此前未知的机制，该机制将启动子基因变异对AQP3表达的影响联系起来，揭示了AQP3在应对PEDV发病机制中的作用，并表明了16 bp插入突变与猪肠道上皮细胞对PEDV感染的抗性之间的潜在关联。
