Brequinar inhibits African swine fever virus replication in vitro by activating ferroptosis

Abstract

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, HAD50, 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.

TRIM21 Promotes Oxidative Stress and Ferroptosis through the SQSTM1-NRF2-KEAP1 Axis to Increase the Titers of H5N1 Highly Pathogenic Avian Influenza Virus

“…Further studies showed that simultaneous inhibition of TRIM21 and NRF2 did not increase the expression of SLC7A11 and GPX4 in A549 cells. These results indicated that knockdown of TRIM21 alleviated ferroptosis by increasing the expressions of NRF2, SLC7A11, and GPX4 in A549 cells infected with H5N1 HPAIV; these findings are consistent with those in the EBV study [56].…”

“…RSL3 is a ferroptosis inducer that has been reported to inhibit the proliferation of porcine epidemic diarrhea virus (PEDV) in Vero cells [ 55 ]. Brequinar activates ferroptosis to inhibit ASFV replication [ 56 ]. However, a variety of viruses can use host ferroptosis to promote their replication, transmission, and pathogenicity [ 28 ].…”

“…These results are consistent with those of NDV, H3N2 influenza virus, and H1N1 influenza virus infection. Knockdown of KEAP1 in nasal epithelial cells increased the expression of NRF2 and enhanced the transcription of SLC7A11 and GPX4 to alleviate ferroptosis induced by H1N1 influenza virus infection [ 56 ]. NRF2 plays an important role in mitigating lipid peroxidation and ferroptosis [ 62 ].…”