The Role Of Autophagy In Replication Of Porcine Parvovirus

Porcine parvovirus(PPV),a member of the autonomous Parvovirus,belonging to the genus Parvovirus of the family Parvoviridae,is a major infectious pathogen of reproductive failure in swine.PPV generally proliferates in porcine-proliferating cells,and the cells infected with PPV produce a significant cytopathic effect(CPE).As an important way to maintain homeostasis in cells,autophagy plays an important role in the pathogenesis of the virus.Although there have been many reports on the mechanism of the interaction between autophagy and viruses,how autophagy regulates the replication of PPV remains unclear.In this study,PK-15 cells were used as model cells,the expression of autophagy-related proteins in PPV infected PK-15 cells and PK-15 cells transfected with GFP-LC3 plasmid was detected by western blotting,and the formation of autophagosomes were observed under fluorescence.PPV infected PK-15 cells,the autophagy model of PK-15 cells was established by autophagy inducer and the PK-15 cells that interfered with the autophagy key gene atg5 using siRNA technology.Then the expression of autophagy-related proteins,PPV VP2 were detected by western blotting,PPV copy number and PPV VP2 mRNA level were detected by Real-time PCR.The study obtained the following results.1.PK-15 cells were infected with 0 MOI,0.25 MOI,0.50 MOI,0.75 MOI,1.0 MOI,and 1.25 MOI PPV for 24 h.The expression of autophagy-related proteins LC3-II and P62 increased with increasing MOI by western blotting.PK-15 cells were infected with 1.0 MOI of PPV at 0 h,6 h,12 h,24 h,36 h,48 h.The expression of autophagy-related proteins LC3-II and P62 expression levels were detected began to increase at 12 h after infection and prolonged with time of infection.PK-15 cells were transfected with GFP-LC3 plasmid for 12 h,then infected these transfected PK-15 cells with 0 MOI,0.75 MOI,1.0 MOI,and 1.25 MOI PPV.At the same time,use the transfected PK-15 cells infected with 1.0 MOI PPV for 0 h,12 h,24 h,36 h.Under the fluorescence microscope,it was found that in the virus gradient group,the autophagosome began to appear at 0.75 MOI,and increased with increasing MOI.In the time gradient group,autophagosomes appeared after 12 h of PPV infection and increased with time.2.Autophagy model was established by EBSS treatment of PK-15 cells for 4 h.Western blotting showed that the expression of LC3-II and P62 in PK-15 cells induced by EBSS increased significantly.Then,EBSS-treated cells were infected with 1.0 MOI of PPV for 24 h.Western blotting showed that the expression of PPV VP2 was significantly increased;total DNA of PK-15 cells was extracted,and the PPV copy number in EBSS-treated cells detected by Real-time PCR was significantly higher than that in the control group(P<0.01);total RNA of PK-15 cells was extracted and reverse-transcribed into cDNA,the expression of PPV VP2 mRNA in EBSS-treated cells detected by Real-time PCR was significantly higher than that in the control group(P<0.01).3.siATG5 interfered with the specific site of autophagy key gene atg5 in PK-15 cells and blocked the activity of atg5 gene.Western blotting results showed that the expression of atg5 gene was inhibited after si ATG5 transfected.Then siATG5 was transfected into EBSS-treated cells.Western blotting showed that the expression of LC3-II decreased and the expression of P62 increased.We utilized 1.0 MOI PPV infected PK-15 cells,siATG5 transfected PK-15 cells,EBSS-treated cells for 24 h,Western blotting results showed that the expression of PPV VP2 in EBSS-treated cells was increased,and the expression of PPV VP2 in siATG5 transfected PK-15 cells was significantly reduced.The total DNA of PK-15 cells was extracted and the results of Real-time PCR showed that the PPV copy number in siATG5 transfected PK-15 cells was significantly lower than that of the control group(P<0.01).Total RNA of PK-15 cells was extracted and reverse-transcribed into cDNA.Real-time PCR showed that the expression of PPV VP2 mRNA in siATG5 transfected PK-15 cells was significantly lower than that in the control group(P<0.01).In this study,we found that PPV infection can induce autophagy in PK-15 cells,and it is clear that autophagy promotes the replication of PPV.Interferes with autophagy key gene atg5 result in inhibition of autophagy,and the replication of PPV is also significantly inhibited.This study revealed the role of autophagy in the promotion of PPV replication and confirmed that autophagy gene atg5 plays a key role in the process of autophagy promotes PPV replication.The research results provide new theoretical data for clarifying the pathogenic mechanism of PPV.