Mechanism Of VP3 Protein Induced Pyroptosis By Chicken Infectious Anemia Virus In HCT116 Cells

Chicken infectious anemia(CIA),also known as blue wing disease or anemia dermatitis syndrome,is an immunosuppressive disease characterized by generalized lymphoid atrophy and a bleeding diathesis in the muscle,which causes huge economic losses to the chicken industry worldwide.CIA is caused by chicken infectious anemia virus(CAV),an icosahedral non enveloped single stranded DNA virus that can cause host infection by vertical and horizontal transmission.The virus has an open reading frame and encodes only three proteins,VP1,VP2,and VP3.The VP3 protein is also known as apoptin because of its proapoptotic activity,and it plays multiple roles in the process of CAV viral infection.In recent years,an increasing number of studies have shown that VP3 proteins are selectively cytotoxic,inducing apoptosis in various transformed cells by aggregation in the nucleus,whereas in normal cells,the localization of VP3 proteins into the cytoplasm leads to inactivation of its proapoptotic activity.The specific mechanism of VP3 which induced cell death is still unclear,and there are no related reports about VP3 protein induced pyroptosis.In this paper,we carried out the following related experiments on the killing effect of VP3protein in cells and its mechanism for that.1.Inhibitory effect of cav-vp3 protein on cell proliferation.In this study,apoptin gene was inserted into them by recombinant adenovirus to exert its biological activity by expressing apoptin after infection of HCT116 cells with recombinant adenovirus.Using crystal violet,CCK-8,and flow cytometry experiments,it is found that cell killing by VP3 was significantly time-and dose-dependent;Through laser confocal microscopy and transmission electron microscopy experiments,the fact is that the cells appeared swollen,holes appeared in the surface of the membrane,and exhibited vesicular like changes,which was quite different from classical apoptosis.Further staining of the nuclei revealed only a small amount of nuclear condensation and nuclear fragmentation,and indicated that only a few cells had undergone apoptosis.Nevertheless,the enlarged and swollen cells with intact nuclei highly resembled the morphological features of pyroptosis,which,combined with the elevated Annexin V~+/PI~+ratio in the flow cytometry results,provided preliminary evidence that VP3 may mediate cell death through the pyroptosis pathway.2.CAV-VP3 proteins induced pyroptosis through the mitochondrial pathway.To reveal the mechanism of VP3 induced pyroptosis,small RNA interference technology was used to knockdown gsdmd and gsdme,respectively,and the results found that the number of pyroptosis like cells did not change significantly after knockdown of GSDMD,and the upstream effector protein of GSDMD,caspase-1,was not activated,while the number of cells with pyroptosis morphology was greatly reduced and the pyroptosis phenomenon was almost completely reversed after knockdown of GSDME,Indicating that VP3 induced pyroptosis is through GSDME and not GSMD.Previous studies have shown that the upstream protein of GSDME is caspase-3,so we determined that caspase-3 was activated by Western blot assay,and found that pyroptosis was almost abolished by knocking down caspase-3,indicating that caspase-3 plays an important role upstream of GSDME.Next,we knocked down caspase-9,bax,Tom20,which are the upstream pathway proteins of caspase-3,and finally determined that VP3was regulated and induced pyroptosis through Tom20-bax-caspase-9-caspase-3-GSDME signaling axis.3.CAV-VP3 proteins activated multiple programmed cell pathways of death.The phenomenon of pyroptosis was almost reversed by knockdown of GSDME,and the survival rate of cells was greatly increased,indicating that VP3 mediated pyroptosis is through GSDME protein and that pyroptosis plays an important role in inhibiting cell proliferation.By detecting the marker proteins of apoptosis and pyroptosis at different time points,VP3 was found to be able to simultaneously induce pyroptosis and the occurrence of apoptosis,and according to GSDME knockdown and overexpression it was found that there was a interconversion of these two programmed cell death ways,that is,apoptosis was inhibited when pyroptosis increased,but increased when pyroptosis decreased.In this study,by staining autophagosomes,autolysosomes,mitochondria,and lysosomes,we found that VP3 induced the activation of autophagy,which was able to upregulate the expression of effector GSDME and decrease cell survival by using autophagy inhibitors and knockdown of ATG5,a key autophagy protein indicated that autophagy plays a protective role in the progression of VP3mediated cell death.4.Verify the mechanism by which VP3 mediates pyroptosis in vivo.In this paper,we established a mouse tumor bearing model by inoculating HCT116 cells in the leg of nude mice and analyzed the samples after VP3 treatment for relevant indicators,and the TUNEL results found that the number of positive cells was low,ie,only a few cells had undergone apoptosis,which was consistent with the previous experimental results.The expressions of pyroptotic proteins(GSDME,caspase-3,caspase-9,bax,cytochrome c,Tom20)were analyzed by Western blot,and the results were consistent with the in vitro results.The present study demonstrated that CAV-VP3 protein similarly significantly inhibited cell proliferation in vivo and also triggered pyroptosis via the mitochondrial pathway.In summary,in this paper,the author found that CAV-VP3 protein activated caspase-3 by activating reactive oxygen species(ROS)signaling to initiate the mitochondrial pathway,which in turn cleaved GSDME to release its N-terminal protein with pore activity to initiate pyroptosis.CAV-VP3 protein can induce the occurrence of both apoptosis and pyroptosis,and transformation occurs under the regulation of GSDME protein,and inhibition of autophagy can further upregulate GSDME-N-terminal protein expression and increase the killing effect of VP3 on cells.The above findings identify a mechanism of pyroptosis induced by CAV-VP3 proteins,refine the mechanism by which VP3 proteins mediate cell death,and lay an important theoretical foundation for further development of VP3 proteins in the future.