Activating Esterase D by PFD5 exerts antiviral effect through inhibiting glutathionization of LAMP1 during Senecavirus A infection

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Seneca virus A (SVA) is a newly discovered small nucleic acid virus, which can cause swine blister disease (PVD). Currently, there is no drug or vaccine. Studies have shown that SVA relies on the endolysosomal pathway to accomplish intracellular transport and release, and can disrupt lysosomal homeostasis, but its specific mechanism has not been revealed. At present, there is limited research on vaccines and antiviral drugs for it. Esterase D (ESD), a serine hydrolase, is active against many substrates and plays a role in inhibiting viral replication. However, the specific mechanism of the thioesterase activity of ESD in the signaling pathway is unknown. In this study, we synthesized and screened a novel small-molecule ESD activator, FPD5 (4-chloro-2-(5-phenyl-1-(pyridin-2-yl)-4,5-dihydro- 1h -pyrazol-3-yl)phenyl), capable of inhibiting the replication of SVA. To explore the mechanism of action of this process, we demonstrated the direct interaction between ESD and lysosomal membrane protein-1 (LAMP1) by CO-IP; Western blot revealed that FPD5 could activate ESD and exert a protective effect on LAMP1 and lysosomes. The deglutathionization function of ESD was verified by protein glutathione immunoblotting and detection reagents, and the affected cysteine residues were found by point mutation technique. The results showed that FPD5 activated ESD to exert thioesterase activity, reduced glutathionylation cysteine 375 in the LAMP1 and decreased SVA production. This study provides a theoretical basis for the development of small molecule drugs against SVA, and also suggests that the activation of the thioesterase activity of ESD is a new direction for future antiviral drug development.