Mechanisms Of Seneca Valley Virus 3C Protein Regulating Selective Autophagy,Stress Granule Formation,and Pyroptosis

Seneca Valley virus(SVV)is an emerging animal virus belonging to the Picornaviridae family.Because SVV has an oncolytic effect on neuroendocrine cancers and is harmless to human,it is considered as an oncolytic virus to treat human cancers.SVV infection can cause vesicular disease(VD)in pigs,and the clinical symptoms are highly similar to foot-and-mouth disease(FMD)and other vesicular diseases.SVV-infected pigs develop anorexia,lethargy,lameness and vesicular lesions.Since the disease was first discovered in Canada in 2007,sporadic epidemics have been reported in major pig-raising provinces in China,posing a potential great threat to the world’s breeding industry.As an emerging infectious disease pathogen in pigs,little is known about the pathogenic characteristics and pathogenic mechanism of SVV.It is urgent to systematically carry out research on its pathogenic characteristics and its pathogenic mechanism,to analyze its pathogenic characteristics,and to clarify its pathogenic mechanism.The development of novel SVV vaccines and the formulation of prevention and control strategies and the transformation of safe and efficient oncolytic viruses provide a scientific basis.Autophagy(autophagy),stress granules(SGs)and pyroptosis are different cellular stress responses in response to various stress stimuli.They play an important role in the development of immune response and disease.This study focused on host-SVV interaction,and systematically explored the molecular mechanisms of SVV infection and how 3C protease regulates selective autophagy,SG formation,and pyroptosis.1.Study on the mechanism of SVV counteracting selective autophagyMacroautophagy/autophagy plays a critical role in antiviral immunity through targeting viruses and initiating host immune responses.We tested the ratio of LC3-I and LC3-II after SVV and UV-inactivated SVV infection.The results showed that SVV infection could induce autophagy,which depends on virus replication.The receptor protein,SQSTM1/p62(sequestosome 1),plays a vital role in selective autophagy.It serves as a receptor targeting ubiquitinated proteins or pathogens to phagophores for degradation.In this study,we explored the reciprocal regulation between selective autophagy receptor SQSTM1 and Seneca Valley virus(SVV).SVV infection induced autophagy.Overexpression of SQSTM1 decreased viral protein production and reduced viral titers.Further study showed that SQSTM1 interacted with SVV VP1 and VP3 independent of its UBA domain.SQSTM1 targeted SVV VP1 and VP3 to phagophores for degradation to inhibit viral replication.To counteract this,SVV evolved strategies to circumvent the host autophagic machinery to promote viral replication.SVV 3C protease targeted the receptor SQSTM1 for cleavage at glutamic acid 355,glutamine 392,and glutamine395 and abolished its capacity to mediate selective autophagy.At the same time,3C-mediated SQSTM1 cleavage products lost the ability to inhibit viral propagation.Collectively,our results provide evidence for selective autophagy in host against viruses and reveal potential viral strategies to evade autophagic machinery for successful pathogenesis.2.Study the mechanism of SVV inhibiting stress granuleStress granules(SGs)are the sites of m RNA storage and related to the regulation of m RNA translation,which are dynamic structures in response to various environmental stresses and viral infections.In this study,we first evaluated whether SVV infection induces SGs formation.Immunofluorescence results showed that50% of cells formed punctate aggregates with the SG marker proteins G3BP1 and e IF4 G after 4 h postinfection(P< 0.01),and then the punctate aggregates gradually decreased as time progressed.SG formation can be induced in the early stage of virus infection and SG formation can be inhibited in the late stage of infection.SVV infection-induced SGs were similar to classical SGs.We further explored the molecular mechanism of SVV infection-induced SG formation.SVV infection induced SG formation in the early stage of infection in a PKR-e IF2α dependent manner.Surprisingly,we found that downregulating SGs marker protein G3BP1 inhibited SG formation,but this inhibition of transient SG formation had no significant effect on SVV propagation.Depletion of G3BP1 significantly attenuated the activation of NF-κB signaling pathway.In addition,we found that SVV inhibited SG formation at the late stage of infection and 3C protease was essential for the inhibition depending on its enzyme activity.Furthermore,we also found that 3C protease blocked the SG formation by disrupting e IF4GI-G3BP1 interaction.3.Study the mechanism of SVV 3C protease inducing pyroptosisPyroptosis is an inflammatory programmed cell death characterized by the formation of pores in the plasma membrane and cell swelling.Through morphological observation and cleavage of porcine-derived GSDMD(p GSDMD),we found that SVV infection could induce pyroptosis in caspase-dependent and-independent ways.Further study showed that 3C protease targeted porcine gasdermin D(p GSDMD)for cleavage through its protease activity.3C protease cleaved porcine GSDMD(p GSDMD)at two sites,glutamine 193(Q193)and glutamine 277(Q277),and Q277 was close to the caspase-1induced p GSDMD cleavage site.p GSDMD1-277 triggered cell death,which was similar to N-terminal fragment produced by caspase-1 cleavage of p GSDMD,and other fragments exhibited no significant inhibitory effects on cellular activity.Ectopic expression of p GSDMD converted 3C-induced apoptosis to pyroptosis in 293 T cells.Interestingly,3C protease did not cleave mouse GSDMD or human GSDMD.Taken together,our results reveal a novel pyroptosis activation manner produced by viral protease cleavage of p GSDMD,which may provide an important insight into the pathogenesis of SVV and cancer therapy.In conclusion,this study systematically evaluated the molecular mechanisms of SVV infection and 3C protease in regulating selective autophagy,stress granule formation,and pyroptosis.This study provides an important insight into SVV-host interaction and the pathogenic mechanism,provides a theoretical basis for the research and development of new SVV vaccines and oncolytic viruses and other biological agents,and also enriches the related research on the regulation of cellular stress responses by picornaviruses.