Molecular Mechanisms Of Cellular Autophagy And Apoptosis Induced By SVV Infection

Seneca Valley virus(SVV)belongs to the genus senecavirus within the family Picornaviridae.In 2002,the virus was discovered in cell culture medium occasionally,and the virus was named SVV-001 firstly.The virus was proposed for the treatment of nervous endocrine cancers as the oncolytic virus infecting cancer cells.In recent years,SVV related to the porcine idiopathic vesicular disease(PIVD)has been found in many countries,including Amercia,Canada,Brazil and so on.SVV infection can cause swine claudication,vesicle,depression and anorexia.The virus mainly leads the death of newborn piglets(<7 days),and the mortality is approximately 30-70%.Autophagy is a cellular physiological process,of degradating and recycling long-lived proteins and organelles,and also plays an important role in maintaining homeostasis in starvation and other cellular stresses.Autophagy is necessary and crucial for resisting the replication of intracellular pathogens.Apoptosis is a self-killing process depending on energy controlled by gene in response to a variety of stimulation,and is very important in maintaining the normal tissue and clearing the old,damaged and infected cells.Viral infection can prevent cell apoptosis from being infected and be conductive to virus persistent infection,to some extent,but also is beneficial to the release of progeny virus by facilitating apoptosis.The purpose of this study is to investigate the relationship between SVV infection and apoptosis and autophagy.The goal is revealing the molecular mechanism of SVV inducing autophagy and apoptosis,and the effect of them on SVV replication.1.The effect of SVV infection on autophagy and apoptosisIn order to evaluate whether SVV infection can induce autophagy and / or apoptosis,293 T cells were infected with SVV,and cells were collected at different time,then the autophagy and apoptosis related proteins were analyzed by western blot.The results showed that SVV infection can promote the conversion of LC3-I to LC3-II.The conversion of LC3-I to LC3-II was increasing with the SVV infection prolonged and the increasing of SVV MOI.The p62 was degradated at 6 hpi.These results suggest that SVV infection can induce autophagy.Under the situation of apoptosis,many kinds of caspase proteins are activated,causing the cleavage of PARP1.The PARP1 was cleavaged after SVV infection at 9 hpi.At the same time a large number of green fluorescence could be observed by fluorescence microscopy via Annexin V-FITC apoptosis kit assay at 9 hpi.These results suggest that SVV infection can induce cell apoptosis.2.The effect of autophagy and apoptosis on the proliferation of SVVSVV infection can induce autophagy effectively.In order to study the effect of autophagy on SVV,we discussed the two aspects of inducing and inhibiting autophagy.293 T cells were treated with 100 n M Rapamycin for 2 h at the first time,then infected with 0.1 MOI SVV.Western blot assay showed the proliferation of SVV was inhibited.After the inhibition of autophagy by autophagy inhibitor 3-MA,the proliferation of SVV was promoted.These results revealed that the autophagy induced by SVV infection is not conducive to viral replication.Similarly,in order to evaluate the apoptosis induced by SVV on the replication of SVV,the different concentration of Z-VAD-FMK adopted,a inhibitor for caspase proteins,is to deal with cells.The results showed that the replication of SVV was inhibited because of the apoptosis inhibited by Z-VAD-FMK.It revealed apoptosis is beneficial for the proliferation of SVV.3.SVV 3C induces the autophagy and apoptosisTo investigate the molecular mechanism of SVV infection inducing autophagy and apoptosis,SVV inactivated by UV infected cells,and results showed that the inactivated SVV couldn’t induce autophagy and apoptosis.It revealed that the autophagy and apoptosis induced by SVV depend on the replication of SVV.The structural and nonstructural proteins of SVV were transfected into 293 T cells respectively,afterwards western blot detected the relevant proteins of autophagy and apoptosis.The results showed that overexpression of 3C protein could induce autophagy and apoptosis.3C protein of the virus in the family of Picornaviridae,is a protease,which plays an important role in viral replication and infection.The 49 histidine of SVV3 C protein(H48)and cysteine at position 160(C160)are two important enzyme active site.The constructions of single point mutation of H48 A and C160 A,and double mutation were transfected into 293 T cells.The results showed that the mutations of 3C protein weakened or completely lost induction of autophagy and apoptosis,suggesting that the 3C protein induces autophagy and apoptosis depending on its protease activity.In brief,this study found that SVV infection can induce autophagy and apoptosis.Autophagy is not conducive to the proliferation of SVV,but apoptosis is favorable for the SVV replication.The 3C of SVV depending on its protease activity induced autophagy and apoptosis.These studies provide experimental datas for the study of pathogenic mechanism of SVV proliferation and infection.