Z-VAD(OMe)-FMK suppresses Seneca Valley Virus replication by targeting the active sites of the 3C protease.

Seneca Valley Virus (SVV) is a picornavirus that causes vesicular lesions in pigs, significantly affecting global swine farming. The SVV 3C protease is essential for processing the viral polyprotein and facilitates immune evasion by cleaving or degrading multiple innate immune proteins. In this study, we identified three caspase inhibitors, including Z-VAD(OMe)-FMK (Z-VAD), Z-FA-FMK (Z-FA), and Z-VDVAD-FMK (Z-VDVAD), which significantly inhibit the cleavage activity of SVV 3C protease using a recombinant protein system. Comparative analysis revealed that Z-VAD exhibited the most potent inhibitory effect in a cell transfection system. Further investigations confirmed that Z-VAD, Z-FA, and Z-VDVAD bound directly to the 3C protein. Molecular docking analysis showed that Z-VAD interacted with key enzymatic site residues His48 and Cys160 of the 3C protease, while Z-VDVAD and Z-FA interacted only with residue Cys160. Infection experiments demonstrated that Z-VAD significantly suppressed the replication by targeting 3C protease. Furthermore, Z-VAD significantly suppressed the replication of Enterovirus A71 (EV-A71) and encephalomyocarditis virus (EMCV). Our findings provide a comprehensive understanding of SVV 3C protease inhibitors and their mechanisms of action, offering valuable insights for the development of strategies to control SVV and other picornaviruses.