Molecular Mechanism Of Porcine Parvovirus Non-structural Protein 1(NS1)-induced Apoptosis Involved In Abortion Of Sows

Porcine parvovirus(PPV)is one of the most important viruses causing reproductive disorder in sows.PPV not only infects the placental tissue of pregnant sows,but also infects the fetus through the placental barrier,causing damage to the placental tissue of sows and fetal development disorder,leading to pregnancy failure and abortion.We and other group have demonstrated that PPV can induce apoptosis and necrosis of host cells,and it is speculated that PPV-induced apoptosis is involved in PPV infection-mediated abortion of pregnant sows.To investigate the molecular mechanism of PPV-induced apoptosis,based on the identification that PPV-induced abortion in sows is related to PPV-induced apoptosis.By focusing on PPV-encoded proteins and apoptosis pathway in host cells,we analyzed and determined the viral protein which mediates PPV-induced apoptosis,and then explored the molecular mechanism of the viral protein-induced apoptosis in host cells.To determine whether PPV-induced abortion in pregnant sows is related to PPV-induced apoptosis and tissue injury,the pathological changes and cell apoptosis of placental tissue from PPV-induced aborted sows were detected by tissue sectioning technique followed by HE or TUNEL staining,respectively.The results showed that PPV infection can cause different degrees of damage of placental tissue,shedding of epithelial cells(including trophoblast cells),hyperemia in the tissues,and severe apoptosis and necrosis of epithelial cells accompanied with tissue damage,suggesting that PPV-induced abortion in pregnant sows is related to PPV-induced cell apoptosis and necrosis.In order to further prove that PPV can induce cell apoptosis,the PPV was used to infect PK-15 host cells in vitro.After infection,flow cytometry and other methods were used to analyze cell apoptosis and morphological changes.The results showed that PPV infection could reduce PK-15 cells survival rate,promote cell genomic DNA fragment and cell membrane phosphatidoserine eversion,and increase Caspase-3 activity,indicating that PPV infection in vitro could induce PK-15 cells apoptosis.The above in vivo and in vitro experiments confirmed that PPV can induce apoptosis of host cells.To investigate the molecular mechanism of apoptosis induced by PPV,it is necessary to determine the viral proteins involved in apoptosis induced by PPV.Therefore,in this study,the molecular cloning technique was used to clone PPV NS1,VP1 and VP2 genes with PCR method,and their eukaryotic expression vectors were constructed.Different expression vectors were transfected into PK-15 cells at different doses for their expression,and the survival rate,phosphatidylserine eversion and Caspase activity of PK-15 cells were detected at the same protein expression level to compare the effects of the three proteins on apoptosis.The results showed that NS1 could significantly promoted cell membrane phosphatidylserine eversion,increased intracellular Caspase-3 activity and decreased cell survival rate,while VP1 and VP2 had no significant effect.Therefore,it was proved that NS1 protein of PPV could induce apoptosis of host cells,indicating that NS1 played an important role in apoptosis induced by PPV.In order to determine the apoptosis pathway in the host cells induced by NS1,NS1 vector was transfected into PK-15 cells,and then the activity of Caspase-3,8 and 9 in the cells was detected by the corresponding kits,and the apoptosis-related proteins were detected by Western blot.The results showed that NS1 could significantly increase the activation of Caspase-9 and Caspase-3(P < 0.05),but had no significant effect on the activity of Caspase-8.NS1 significantly promoted the expression of pro-apoptosis-related proteins(Bax,p21 and p53),while inhibited the expression of anti-apoptosis-related proteins(Bcl-2 and Mcl-1).Meanwhile,NS1 also significantly promoted the release of mitochondrial CytC.However,NS1 had no significant effect on the expression of proteins related to the external pathway of apoptosis(such as Fas,FasL and TRAIL).It can be seen that NS1 induces cell apoptosis through the internal mitochondrial pathway,rather than the external Fas/FasL-mediated death receptor pathway.To know how NS1 induces apoptosis through the mitochondrial pathway,NS1 vectors were transfected into PK-15 cells,and the levels of phosphatidylserine eversion and mitochondrial ROS in PK-15 cells were detected by flow cytometry and other techniques.Mitochondrial damage was detected by fluorescein labeling/flow cytometry.TMRM fluorescein/ flow cytometry was used to detect mitochondrial membrane potential(? ? m).Results show that the NS1 could obviously increase the levels of ROS in cells(P < 0.01),and promote the mitochondrial damage and lower mitochondrial ? ? m,NAC a ROS inhibitor,could inhibit the NS1-induced apoptosis,ROS production,mitochondrial injury and mitochondrial ? ? m lost.These results suggest that NS1-induced apoptosis is closely related to mitochondrial damage induced by ROS production.To further investigate the role of ROS in NS1-induced apoptosis,the effects of NS1 on cell DNA damage and cell cycle were detected by Western blot and flow cytometry,respectively.The results showed that NS1 significantly increased the expression of ?-H2 AX,indicating that NS1 can cause damage to cell DNA,affect the normal cell cycle,and make the cell cycle arrested at G1/G2 phases.ROS inhibitor NAC partially reverses these NS1-mediated effects,reducing the expression of ?-H2 AX and the number of G1/G2 cells.Therefore,ROS plays an important role in NS1-mediated cell DNA damage and cell cycle arrest.To sum up,based on proving that PPV infection can induce apoptosis,cause damage to placental tissue,and cause abortion of pregnant sows,we mainly explored the viral protein which mediating PPV-induced apoptosis and the molecular mechanism of viral protein-induced apoptosis.We first found that PPV-induced apoptosis is closely related to the PPV-encoded non-structural protein 1(NS1).NS1 induces apoptosis of host cells by activating mitochondrial pathway,and ROS plays an important role in NS1-induced apoptosis.This study provides an important experimental basis for exploring the molecular mechanism of PPV-induced apoptosis and tissue injury resulting in abortion in pregnant sows.