线粒体是细胞中的能量生产者,可以通过调节宿主先天免疫信号通路来影响病毒复制,它们的生物学功能的变化与病毒的生命周期密不可分。在这项研究中,我们筛选了382个线粒体靶向化合物的文库,并鉴定了二氢乳清酸脱氢酶(DHODH)的抗病毒抑制剂,嘧啶核糖核苷酸从头合成途径中的限速酶,针对经典猪瘟病毒(CSFV)。我们的数据显示,这些抑制剂以剂量依赖的方式干扰病毒RNA合成,半最大有效浓度(EC50)范围为0.975至26.635nM。值得注意的是,DHODH抑制剂通过增强包括TBK1-IRF3-STAT1和NF-κB信号通路在内的先天免疫应答来阻断CSFV复制。此外,来自一系列化合物添加和补充试验的数据表明,DHODH抑制剂还通过阻断嘧啶从头合成来抑制CSFV复制.值得注意的是,DHODH敲除表明其对于CSFV复制是必需的。机械上,共聚焦显微镜和免疫沉淀分析显示,非结构蛋白4A(NS4A)在核周募集并与DHODH相互作用。值得注意的是,NS4A增强了DHODH活性并促进了UMP的产生,以实现有效的病毒复制。在结构上,DHODH的氨基酸65-229和NS4A的氨基酸25-40是这种相互作用的关键。一起来看,我们的研究结果强调了DHODH在CSFV生命周期中的关键作用,并为开发抗CSF新疗法提供了潜在的抗病毒靶点.
目的:猪瘟仍然是世界范围内最重要的家猪和野猪病毒性疾病之一。二氢乳清酸脱氢酶(DHODH)抑制剂已被证明可以在体外和体内抑制几种病毒的复制,但对瘟病毒的影响仍然未知。在这项研究中,三种特异性DHODH抑制剂,包括DHODH-IN-16,BAY-2402234和Brequinar被发现强烈抑制经典猪瘟病毒(CSFV)的复制。这些抑制剂靶向宿主DHODH,耗尽嘧啶核苷酸库以发挥其抗病毒作用。有趣的是,我们观察到CSFV的非结构蛋白4A诱导DHODH与线粒体一起在细胞核周围积累。此外,NS4A与DHODH表现出强烈的相互作用,增强其活性以促进高效的CSFV复制。总之,我们的发现增强了对CSFV感染中嘧啶合成的理解,并扩展了CSFVNS4A在病毒复制中的新功能,为进一步探索抗CSFV的抗病毒靶点提供参考。
Mitochondria are energy producers in cells, which can affect viral replication by regulating the host innate immune signaling pathways, and the changes in their biological functions are inextricably linked the viral life cycle. In this study, we screened a library of 382 mitochondria-targeted compounds and identified the antiviral inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo synthesis pathway of pyrimidine ribonucleotides, against classical swine fever virus (CSFV). Our data showed that the inhibitors interfered with viral RNA synthesis in a dose-dependent manner, with half-maximal effective concentrations (EC50) ranging from 0.975 to 26.635 nM. Remarkably, DHODH inhibitors obstructed CSFV replication by enhancing the innate immune response including the TBK1-IRF3-STAT1 and NF-κB signaling pathways. Furthermore, the data from a series of compound addition and supplementation trials indicated that DHODH inhibitors also inhibited CSFV replication by blocking the de novo pyrimidine synthesis. Remarkably, DHODH knockdown demonstrated that it was essential for CSFV replication. Mechanistically, confocal microscopy and
immunoprecipitation assays showed that the non-structural protein 4A (NS4A) recruited and interacted with DHODH in the perinuclear. Notably, NS4A enhanced the DHODH activity and promoted the generation of UMP for efficient viral replication. Structurally, the amino acids 65-229 of DHODH and the amino acids 25-40 of NS4A were pivotal for this interaction. Taken together, our findings highlight the critical role of DHODH in the CSFV life cycle and offer a potential antiviral target for the development of novel therapeutics against CSF.
OBJECTIVE: Classical swine fever remains one of the most economically important viral diseases of domestic pigs and wild boar worldwide. dihydroorotate dehydrogenase (DHODH) inhibitors have been shown to suppress the replication of several viruses in vitro and in vivo, but the effects on Pestivirus remain unknown. In this study, three specific DHODH inhibitors, including DHODH-IN-16, BAY-2402234, and Brequinar were found to strongly suppress classical swine fever virus (CSFV) replication. These inhibitors target the host DHODH, depleting the pyrimidine nucleotide pool to exert their antiviral effects. Intriguingly, we observed that the non-structural protein 4A of CSFV induced DHODH to accumulate around the nucleus in conjunction with mitochondria. Moreover, NS4A exhibited a strong interaction with DHODH, enhancing its activity to promote efficient CSFV replication. In conclusion, our findings enhance the understanding of the pyrimidine synthesis in CSFV infection and expand the novel functions of CSFV NS4A in viral replication, providing a reference for further exploration of antiviral targets against CSFV.