Molecular Mechanisms Of Senecavirus A(SVA)VP1 Interacted With Host BAD To Activate Mitochondrial Apoptosis Pathway

Apoptosis refers to the process in which the organism commits suicide by regulating genes in order to maintain homeostasis.Apoptosis is one of the first lines of defense against pathogen invasion and replication.Virus acts as a strictly parasitic pathogen whose survival and expansion must depend on the host.Apoptosis occurred prematurely can cause the progeny virus phagocytosed and excluded by the immune surveillance and defense system.Therefore,many viruses inhibit cell death through a mechanism to assure survive stably in the host cell.Conversely,when the progeny virions complete the assembly,the virus will utilize the pro-apoptotic mechanism of the host cell to promote apoptosis,and spread the progeny virus to achieve the purpose of persistent infection.Senecavirus A(SVA)is a small,non-enveloped,single-stranded RNA virus in the family Picornaviridae.In 2002,SVA was first identified in the cell culture contaminant for culturing human fetal retinal cells.As a susceptible animal of SVA,pigs may have intact or broken vesicles in the nose or oral mucosa of the pig after infection with SVA.Incompleteness of the mucous membrane in the hoof leads to lameness In addition,SVA infection can also lead to acute death of newborn piglets.SVA is a new class of pathogens of the picornavirus family.The relationship between it infection and host cell apoptosis has not yet been elucidated.By HE staining and paraffin section fluorescent TUNEL experiments,it was found that SVA can cause obvious pathological changes in host tissues and produce obvious apoptosis.In order to explore its effect on host cell apoptosis,we first observed that SVA infection can cause significant cytopathic effects in PK-15 and HEK293 T cells through microscope.By qPCR assay and flow cytometry,it was found that SVA can replicate in 293 T cells,and it also can induce significant apoptosis in 293 T cells compared to the control.The results of Western Blotting and flow cytometry showed that SVA structural protein VP1 could induce apoptosis in a dose-dependent manner.Aim to research the molecular mechanism of apoptosis induced by VP1,we observed fluorescence staining by Annexin-V-FITC/PI,and found that VP1 can induce apoptosis in early and late stages.Co-immunoprecipitation,indirect immunofluorescence and mitochondrial separation experiments revealed that VP1 localizes on mitochondria and interacts with the pro-apoptotic protein BAD.Overexpression of BAD could promote apoptosis induced by VP1.However,the function of VP1 to induce apoptosis was significantly reduced after siRNA knockdown of BAD.Western Blotting and qPCR assays showed that VP1 could promote the expression of BAX and the cleavage activation of Caspase3 in the downstream of BAD,but had no effect on the upstream proteins AKT,JNK and their phosphorylation.After BAD interference,the promotion of BAX and Caspase3 by VP1 disappeared,indicating that VP1 is dependent on BAD to activate the mitochondrial apoptosis pathway.It was also found by qPCR that BAD can promote viral replication in the late stage of SVA-infected cells.Therefore,it indicated that SVA-VP1 directly interacts with BAD on the mitochondria,thereby activating BAX which in turn activates Caspase3 and ultimately causes apoptosis.This study is the first to elucidate the effect of SVA structural protein VP1 on apoptosis in 293 T cells,which helps to reveal the molecular mechanism by which SVA utilizes host proteins to influence host cell apoptosis and self-replication,and provides a theoretical basis for further revealing the pathogenic mechanism of SVA.