布喹那抑制非洲猪瘟病毒在体外的复制是通过激活铁死亡。
Brequinar inhibits African swine fever virus replication in vitro by activating ferroptosis.
机构信息
Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
African Swine Fever Regional Laboratory of China (Guangzhou), Guangzhou, China.
出版信息
Virol J. 2023 Oct 24;20(1):242. doi: 10.1186/s12985-023-02204-x.
BACKGROUND
African swine fever virus (ASFV) is one of the most fatal swine etiological agents and has a huge economic impact on the global pork industry. Given that no effective vaccines or anti-ASFV drugs are available, there remains a pressing need for novel anti-ASFV drugs. This study aimed to investigate the anti-African swine fever virus (ASFV) activity of brequinar, a DHODH inhibitor.
METHODS
The anti-ASFV activity of brequinar was investigated using IFA, HAD, HAD, qRT-PCR, and western blotting assays. The western blotting assay was used to investigate whether brequinar inhibits ASFV replication by killing ASFV particles directly or by acting on cell factors. The confocal microscopy and western blotting assays were used to investigate whether brequinar inhibits ASFV replication by activating ferroptosis.
RESULTS
In this study, brequinar was found to effectively inhibit ASFV replication ex vivo in porcine alveolar macrophages (PAMs) in a dose-dependent manner. In kinetic studies, brequinar was found to maintain ASFV inhibition from 24 to 72 hpi. Mechanistically, the time-of-addition assay showed that brequinar exerted anti-ASFV activity in all treatment modes, including pre-, co-, and post-treatment rather than directly killing ASFV particles. Notably, FerroOrange, Mito-FerroGreen, and Liperfluo staining experiments showed that brequinar increased the accumulation of intracellular iron, mitochondrial iron, and lipid peroxides, respectively. Furthermore, we also found that ferroptosis agonist cisplatin treatment inhibited ASFV replication in a dose-dependent manner and the inhibitory effect of brequinar on ASFV was partially reversed by the ferroptosis inhibitor ferrostatin-1, suggesting that brequinar activates ferroptosis to inhibit ASFV replication. Interestingly, exogenous uridine supplementation attenuated the anti-ASFV activity of brequinar, indicating that brequinar inhibits ASFV replication by inhibiting DHODH activity and the depletion of intracellular pyrimidine pools; however, the induction of ferroptosis by brequinar treatment was not reversed by exogenous uridine supplementation, suggesting that brequinar activation of ferroptosis is not related to the metabolic function of pyrimidines.
CONCLUSIONS
Our data confirm that brequinar displays potent antiviral activity against ASFV in vitro and reveal the mechanism by which brequinar inhibits ASFV replication by activating ferroptosis, independent of inhibiting pyrimidine synthesis, providing novel targets for the development of anti-ASFV drugs.
背景
非洲猪瘟病毒（ASFV）是最致命的猪病病原体之一，对全球猪肉产业造成了巨大的经济影响。鉴于目前尚无有效的疫苗或抗 ASFV 药物，因此仍然迫切需要新型抗 ASFV 药物。本研究旨在探讨 DHODH 抑制剂布雷喹纳对非洲猪瘟病毒（ASFV）的抗活性。
方法
通过 IFA、HAD、qRT-PCR 和 Western blot 实验检测布雷喹纳的抗 ASFV 活性。Western blot 实验用于研究布雷喹纳是否通过直接杀死 ASFV 颗粒或作用于细胞因子来抑制 ASFV 复制。共聚焦显微镜和 Western blot 实验用于研究布雷喹纳是否通过激活铁死亡来抑制 ASFV 复制。
结果
本研究发现，布雷喹纳可在体外剂量依赖性方式有效抑制猪肺泡巨噬细胞（PAMs）中 ASFV 的复制。在动力学研究中，发现布雷喹纳可维持 ASFV 抑制作用，从 24 小时到 72 小时感染后（hpi）。作用机制研究表明，时间添加实验表明，布雷喹纳在所有处理模式下（包括预处理、共处理和后处理）都发挥了抗 ASFV 活性，而不是直接杀死 ASFV 颗粒。值得注意的是，FerroOrange、Mito-FerroGreen 和 Liperfluo 染色实验表明，布雷喹纳分别增加了细胞内铁、线粒体铁和脂质过氧化物的积累。此外，我们还发现，铁死亡激动剂顺铂处理可剂量依赖性地抑制 ASFV 复制，而铁死亡抑制剂 ferrostatin-1 部分逆转了布雷喹纳对 ASFV 的抑制作用，表明布雷喹纳通过激活铁死亡来抑制 ASFV 复制。有趣的是，外源性尿嘧啶补充剂减弱了布雷喹纳的抗 ASFV 活性，表明布雷喹纳通过抑制 DHODH 活性和耗尽细胞内嘧啶池来抑制 ASFV 复制；然而，外源性尿嘧啶补充剂并未逆转布雷喹纳处理诱导的铁死亡，表明布雷喹纳激活铁死亡与嘧啶代谢功能无关。
结论
本研究数据证实，布雷喹纳在体外对 ASFV 表现出强大的抗病毒活性，并揭示了布雷喹纳通过激活铁死亡抑制 ASFV 复制的机制，与抑制嘧啶合成无关，为开发抗 ASFV 药物提供了新的靶点。
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