Seneca Valley Virus 3C Protease Induces Pyroptosis by Directly Cleaving Porcine Gasdermin D.

Seneca Valley virus (SVV), a newly emerging virus belonging to the Picornaviridae family, has caused vesicular disease in the swine industry. However, the molecular mechanism of viral pathogenesis remains poorly understood. This study revealed that SVV infection could induce pyroptosis in SK6 cells in a caspase-dependent and -independent manner. SVV may inhibit caspase-1 activation at late infection because of 3Cpro cleavage of NLRP3, which counteracted pyroptosis activation. Further study showed that 3Cpro targeted porcine gasdermin D (pGSDMD) for cleavage through its protease activity. 3Cpro cleaved porcine GSDMD (pGSDMD) at two sites, glutamine 193 (Q193) and glutamine 277 (Q277), and Q277 was close to the caspase-1-induced pGSDMD cleavage site. pGSDMD1-277 triggered cell death, which was similar to N-terminal fragment produced by caspase-1 cleavage of pGSDMD, and other fragments exhibited no significant inhibitory effects on cellular activity. Ectopic expression of pGSDMD converted 3Cpro-induced apoptosis to pyroptosis in 293T cells. Interestingly, 3Cpro did not cleave mouse GSDMD or human GSDMD. And, both pGSDMD and pGSDMD1-277 exhibited bactericidal activities in vivo. Nevertheless, pGSDMD cannot kill bacteria in vitro. Taken together, our results reveal a novel pyroptosis activation manner produced by viral protease cleavage of pGSDMD, which may provide an important insight into the pathogenesis of SVV and cancer therapy.

Removal of 3C protease from the 3ABC improves expression, solubility, and purification of the recombinant 3AB of foot-and-mouth disease virus.

During an ongoing outbreak of Foot-and-Mouth Disease Virus (FMDV), it is crucial to distinguish naturally infected from vaccinated seropositive animals. This would support clinical assessment and punctual vigilance. Assays based on 3ABC non-structural protein as an antigen are reliable for this intention. However, the insolubility and degradation of recombinant 3ABC during expression and purification are serious challenges. In this study, alternatively to expressing the recombinant 3ABC (r3ABC), we expressed the 3AB coding sequence (~672 bp) as a recombinant protein (r3AB) with a molecular mass of ~26 KDa. Analytical data from three-dimensional structure, hydrophilicity, and antigenic properties for 3ABC and 3AB exhibited the 3C protein as a hydrophobic, while 3AB as a hydrophilic and highly antigenic protein. The expressed r3AB was recovered as a completely soluble matter after merely native purification, unlike the full expressed r3ABC. Immunoreactivity of r3AB to anti-FMDV antibody in infected sera with different FMDV serotypes was confirmed by the western blot and indirect ELISA. Besides, the authentic antigenicity of purified r3AB was demonstrated through its ability to induce specific seroconversion in mice. Summarily, the removal of 3C: has influenced neither 3D structure nor antigenic properties of the purified r3AB, overcame insolubility and degradation of the r3ABC, and generated a potential superior antigen (r3AB) for herd screening of animals to any FMDV serotype.