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.
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Background
African swine fever (ASF) is a major economically important infectious disease caused by the African swine fever virus (ASFV), which threatens the global pork industry and has high mortality rate [1]. In the 1920s, ASF was first reported in Kenya and was limited to Africa [2]. In the 1950s, it spread to Europe, including Spain, Portugal, Italy, and France [3]. Europe (except for Sardinia) has eradicated ASFV using drastic control and eradication programs. Unfortunately, the disease reemerged in the Caucasus region in 2007 and rapidly spread to the eastern territory of the European Union in 2014 [4]. Next, the disease was reported on August 3, 2018, in China, one of the largest pork industries in the world. Between 2018 and 2022, 204 ASF outbreaks across 32 Chinese provinces were reported by the Chinese Ministry of Agriculture and Rural Affairs, causing huge economic losses, with estimates of at least 1.2 million sick and culled pigs [5, 6].
ASFV is the only member of the Asfarviridae family. It is a large, enveloped, double-stranded DNA virus, with 151–167 open reading frames, encoding more than 150 proteins [7]. ASFV is highly restricted to macrophages and monocytes, especially porcine alveolar macrophages (PAMs), which are the primary targets of ASFV in vivo [8]. Owing to the limited cell tropism and complex viral particle structure of ASFV, research on ASFV is exceedingly difficult, and there are no commercial vaccines or drugs to control ASFV infection [9]. So far, the main strategy to control ASF includes disinfection of vehicles and transit areas, strengthening of biosafety management on pig farms, and stricter vigilance programs [37]. In this context, we attempted to develop an antiviral drug therapy against ASFV. The DHODH inhibitor brequinar is a broad-spectrum antiviral inhibitor, we explored whether brequinar possesses anti-ASFV activity.
In this study, we demonstrated that brequinar strongly inhibits ASFV in a dose-dependent manner (Figs. 2 and 3) and sustain inhibition of ASFV from 24 to 72 hpi (Fig. 3). In general, the compounds exhibit antiviral activity in two ways: either the compound directly targets the virus itself, or the compound impairs host cell factors that are essential for the viral life cycle [38]. Therefore, we investigate whether brequinar interacts directly with ASFV thereby killing ASFV particles. However, the results showed that brequinar did not directly interact with ASFV; instead, brequinar inhibited ASFV replication in different treatment modes, including pre-, co-, and post-treatment, suggesting that brequinar inhibited ASFV replication by acting on cell factors (Fig. 4). DHODH, the fourth enzyme in the de novo pyrimidine biosynthesis pathway, is a popular target for antiviral and anticancer activities. Uridine is the precursor of the pyrimidine nucleotide. Exogenous uridine can be taken up by human ovarian cancer cell line 2008 cells and the uptake rate is essentially linear during the first 30 min [39]. Additionally, exogenous uridine has been used to supplement the consumption of endogenous uridine by the drugs in vitro and in vivo studies [40, 41]. As previously mentioned, brequinar exerted a broad-spectrum antiviral activity by inhibiting DHODH activity and depleting intracellular pyrimidine pools. Consistent with the reported results, we also found that exogenous supplementation with pyrimidines reversed the anti-ASFV activity of brequinar, demonstrating that brequinar suppressed ASFV replication by inhibiting DHODH activity and depleting intracellular pyrimidine pools (Fig. 5).
DHODH is thought to be an enzyme required for the de novo synthesis of pyrimidine nucleotides, but recently Mao et al. found that brequinar activates ferroptosis by inhibiting DHODH activity independently of GPX4 or FSP1 [30]. Ferroptosis is a recently discovered form of cell death characterized by massive iron accumulation and lipid peroxidation [42]. The virus-induced cell death has long been recognized as a double-edged sword that inhibits or exacerbates viral replication [43]. Cheng et al. found that SIV promotes viral replication by activating GPX4-mediated ferroptosis [44]. However, the effect of ASFV infection on ferroptosis and whether brequinar inhibits ASFV replication through ferroptosis are still unknown. Therefore, we investigated the impact of ASFV infection on ferroptosis and the results showed that ASFV infection did not induce ferroptosis; however, brequinar treatment induced ferroptosis and the accumulation of intracellular Fe2+, mitochondrial Fe2+ or lipid peroxides, which were not reversed by exogenous uridine supplementation. We further explored the effect of ferroptosis on ASFV replication and, as expected, treatment with the ferroptosis agonist cisplatin inhibited ASFV replication and found that the inhibitory effect of brequinar on ASFV was partially reversed by the ferroptosis inhibitor ferrostatin-1 (Fig. 5). Hence, the ferroptosis pathway may be a target for the development of anti-ASFV compounds.
It is important to note that there were some side effects with brequinar. Thrombocytopenia was the main side effect, but it was dose-limiting [45]. Given that all drugs have side effects, these clinical observations are acceptable. Importantly, the selectivity index > 20 of brequinar and the effective concentration of brequinar used in our study are much lower than the doses used in previous clinical trials [46]. Although inhibitors can negatively affect cellular function and may lead to deleterious long-term and broad consequences by targeting host cell signaling pathways, the safety and pharmacology of brequinar have already been tested in clinical trials [47, 48]. Moreover, Li and colleagues showed that brequinar inhibited FMDV replication and provided a 25% survival rate in FMDV-infected mice in vivo, suggesting that brequinar could be an effective anti-FMD antiviral agent [27]. Therefore, develo** brequinar as an anti-ASFV inhibitor has advantages over develo** new drugs or identifying new antiviral strategies.
Conclusions
In summary, 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. Therefore, brequinar has potential as a novel drug or adjuvant therapeutic option to combat ASFV infection, and the ferroptosis pathway can be used as a novel target for anti-ASFV drug development.
Data Availability
The datasets used and/or analyzed in this study are obtained and available from the corresponding authors upon a reasonable request.
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Acknowledgements
We would like to thank Editage (www.editage.cn) for English language editing.
Funding
This research was funded by the Guangzhou Basic and Applied Basic Research Foundation (202201010490), Science and Technology Program of Guangzhou, China (202206010036), Start-up Research Project of Maoming Laboratory (2021TDQD002), and China Agriculture Research System of MOF and MARA (cars-35).
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Y.S. and G.Z. designed the study and revised the manuscript. Y.C. per-formed the experiments and wrote the paper. Y.G. and H.C. performed the experiments. Z.S., Z.H. and Z.W. participated in the establishment of the methodology and data analysis. Z.W. and Z.Z. provided some critical reagents. All authors have read and agreed to the published version of the manuscript.
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Chen, Y., Guo, Y., Chang, H. et al. Brequinar inhibits African swine fever virus replication in vitro by activating ferroptosis. Virol J 20, 242 (2023). https://doi.org/10.1186/s12985-023-02204-x
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DOI: https://doi.org/10.1186/s12985-023-02204-x