Yang Li, Du Xiaomei, Hu Yueqing, Wu Zhengchang, Bao Wenbin
College of Animal Science and Technology, Yangzhou University, Yangzhou, PR China.
College of Animal Science and Technology, Yangzhou University, Yangzhou, PR China.
Int J Biol Macromol. 2025 May;310(Pt 3):143501. doi: 10.1016/j.ijbiomac.2025.143501. Epub 2025 Apr 24.
Porcine epidemic diarrhea (PED), induced by porcine epidemic diarrhea virus (PEDV) infection, represents a highly contagious swine disease that causes substantial economic losses in the global pig industry. As the most prevalent RNA modification, N6-methyladenosine (mA) has emerged as a crucial epitranscriptomic regulator of host-pathogen interactions, although its functional significance in PEDV-infected porcine intestinal epithelial cells (IPEC-J2) remains to be fully elucidated. This study drew m6A maps before and after ALKBH5 interfering with IPEC-J2 cells, revealing the mechanism by which ALKBH5 mediated mA regulates PEDV infection in IPEC-J2 cells. This study employs an in vitro model of PEDV-induced IPEC-J2 cell damage to investigate the regulatory mechanisms of m6A methylation in host antiviral responses. Utilizing a comprehensive multi-omics approach, including MeRIP-seq, RNA-seq, qPCR, Western blot, MeRIP-qPCR, and RIP-qPCR, we systematically uncovered the critical role of m6A methylation in antiviral defense. Our findings reveal that ALKBH5, a key demethylase during PEDV infection, plays a pivotal role in modulating m6A modification levels. Specifically, gene silencing of ALKBH5 significantly upregulates m6A modification but downregulates expression of the antiviral effector genes IFIT3 and HERC5. Mechanistically, we demonstrate that the YTHDC2 protein selectively recognizes mA modification sites within IFIT3 and HERC5 transcripts, thereby mediating ALKBH5-dependent regulation of mRNA stability and protein expression. Functional analyses further reveal that IFIT3 activates the IRF3/TBK1 signaling axis, while HERC5 enhances antiviral responses by modulating the key effector molecule ISG15. Together, these findings establish a synergistic antiviral mechanism in IPEC-J2 cells. This study is the first to elucidate the novel ALKBH5-YTHDC2-m6A molecular axis, which orchestrates host antiviral immunity through a dual-pathway regulatory mode. These findings provide a theoretical foundation for understanding the role of RNA epigenetic modifications in enterovirus infections and offer a molecular basis for developing m6A-targeted strategies to prevent and control porcine infectious gastroenteritis.
猪流行性腹泻（PED）由猪流行性腹泻病毒（PEDV）感染引起，是一种高度传染性的猪病，给全球养猪业造成了巨大经济损失。作为最普遍的RNA修饰，N6-甲基腺苷（m6A）已成为宿主-病原体相互作用的关键表观转录组调节因子，尽管其在PEDV感染的猪肠上皮细胞（IPEC-J2）中的功能意义仍有待充分阐明。本研究绘制了ALKBH5干扰IPEC-J2细胞前后的m6A图谱，揭示了ALKBH5介导的m6A调节IPEC-J2细胞中PEDV感染的机制。本研究采用PEDV诱导的IPEC-J2细胞损伤的体外模型，研究m6A甲基化在宿主抗病毒反应中的调节机制。利用包括MeRIP-seq、RNA-seq、qPCR、Western blot、MeRIP-qPCR和RIP-qPCR在内的综合多组学方法，我们系统地揭示了m6A甲基化在抗病毒防御中的关键作用。我们的研究结果表明，ALKBH5是PEDV感染期间的关键去甲基化酶，在调节m6A修饰水平方面发挥着关键作用。具体而言，ALKBH5基因沉默显著上调m6A修饰，但下调抗病毒效应基因IFIT3和HERC5的表达。从机制上讲，我们证明YTHDC2蛋白选择性识别IFIT3和HERC5转录本中的m6A修饰位点，从而介导ALKBH5依赖的mRNA稳定性和蛋白质表达调节。功能分析进一步表明，IFIT3激活IRF3/TBK1信号轴，而HERC5通过调节关键效应分子ISG15增强抗病毒反应。这些发现共同在IPEC-J2细胞中建立了一种协同抗病毒机制。本研究首次阐明了新型的ALKBH5-YTHDC2-m6A分子轴，该分子轴通过双途径调节模式协调宿主抗病毒免疫。这些发现为理解RNA表观遗传修饰在肠道病毒感染中的作用提供了理论基础，并为开发针对m6A的策略以预防和控制猪传染性胃肠炎提供了分子依据。
