Classical swine fever virus non-structural protein 4A recruits dihydroorotate dehydrogenase to facilitate viral replication.

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. IMPORTANCE: 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.

HcFAR, a functional inhibitor of goat TGF-ß1 identified from excretory and secretory products of Haemonchus contortus.

Haemonchus contortus has developed complexed and multifaceted mechanisms of immune evasion to enable the survival in the host. Generating excretion and secretion products (ESPs) to subvert or suppress the functions of host cytokines is a newly immune regulatory pattern found during recent years. Transforming growth factor-ß (TGF-ß) has critical immune regulatory functions in nematode infections. In this study, co-immunoprecipitation (co-IP) assay was used to identify the goat TGF-ß1 binding proteins from HcESPs. The interaction between TGF-ß1 and nematode fatty acid retinoid binding domain containing protein of H. contortus (HcFAR) was analyzed by glutathione S-transferase (GST)-pull down assay. The suppressive effect of rHcFAR on TGF-ß1-induced immunoglobulin A (IgA) secretion was observed by co-incubation of rHcFAR and TGF-ß1 with goat peripheral blood mononuclear cells (PBMCs). The IgA concentrations were determined using enzyme linked immunosorbent assay (ELISA) kit. Meanwhile, the suppressive effect of rHcFAR on TGF-ß1-induced T helper (Th) 9 differentiation was investigated by co-incubation of rHcFAR, TGF-ß1 and interleukin (IL)-4 with goats PBMCs. In parallel, IL-4 was replaced by IL-6 to determine the effects on the Th17 differentiation. The transcriptions of IL-9 and IL-17 in PBMCs were then evaluated by real-time PCR. Finally, we found that HcFAR from HcESPs could bind to goat TGF-ß1 in vitro. The ELISA results of IgA showed that 40 µg/mL rHcFAR could suppress the IgA secretion of PBMCs induced by TGF-ß1. Additionally, rHcFAR (at 10 µg/mL and 20 µg/mL) could inhibit the mRNA transcription of IL-9 induced by TGF-ß1 and IL-4. Meanwhile, rHcFAR could also downregulate the transcription of IL-17 induced by TGF-ß1 and IL-6 in a dose-dependent manner. These results indicated that HcFAR was a functional inhibitor of goat TGF-ß1 and this information may help contribute to understanding of the relationship between the ESPs and host cytokines.