Molecular Mechanism Of Porcine Circovirus Type 3 Cap Protein Activating Mitochondrial Apoptosis Pathway

Porcine Circovrius Type 3(PCV3)is a new type of porcine circovirus found in the United States in 2016.It is prevalent in many countries worldwide and is increasing year by year.At present,studies have shown that PCV3 infection is mainly associated with clinical symptoms such as dermatitis,nephrotic syndrome and reproductive disorders,but the pathogenesis of PCV3 is still unclear.Capsid protein,as the only structural protein of PCV3,plays an important role in the interaction with host cytokines,virus infection or replication.Therefore,in-depth study on the function of Cap protein in PCV3 cell infection and molecular pathogenicity will help us better understand the virus characteristics and pathogenicity of PCV3,and lay a theoretical foundation for the subsequent PCV3 prevention and vaccine research.Therefore,this study carried out the following work:1.Study on the mechanism of apoptosis induced by PCV3 Cap proteinIn this study,p CI-neo-PCV3 Cap eukaryotic expression plasmid was constructed and transfected into PK-15 cells.The activation of apoptosis pathway-related proteins was detected by Western blot.The results showed that with the increase of Cap expression time,the cleavage levels of Caspase-3 and PARP proteins were significantly increased,and Caspase-9,which was involved in mitochondrial apoptosis pathway,was activated,while Caspase-8 did not change significantly.JC-1 fluorescent dye was used to detect the effect of Cap protein on mitochondrial membrane potential.The results showed that Cap protein induced the decrease of cell membrane potential and the increase of mitochondrial membrane permeability.Western blot was used to detect the release of cytochrome c(Cyt c)and apoptosis inducing factor(AIF)related to the mitochondrial apoptosis pathway induced by Cap protein.The results showed that Cyt c was released from mitochondria to cytoplasm,but no AIF was detected in the cytoplasm.The above results further confirmed that PCV3 Cap protein overexpression could induce mitochondrial apoptosis,and only activated Caspase-dependent mitochondrial apoptosis pathway.2.Screening of potential host interacting proteins of PCV3 Cap protein by GST-pull down combined mass spectrometryExploring the interaction between viral proteins and host factors is helpful to understand the pathogenesis of virus.The interaction proteins(bands)of PCV3 Cap protein in PK-15 cells were screened by GST-pull down test and electrophoresis silver staining analysis.The differential proteins were sequenced by liquid chromatographymass spectrometry(LC-MS),and 58 differential proteins were identified.Four candidate proteins(DDX1,G3BP1,hnRNPK and HSP90AB1)were selected for indirect immunofluorescence(IFA)and immune co-precipitation(Co-IP)test verification,and the results showed that : PCV3 Cap protein had intracellular interactions with hnRNPK and G3BP1 proteins,but no intracellular interactions with DDX1 and HSP90AB1.3.Preliminary study of host protein hnRNP K inhibiting mitochondrial apoptosis induced by Cap proteinIn order to investigate the relationship between hnRNP K and apoptosis induced by Cap protein,Western blot was used to detect the protein samples(different time points and different doses)after Cap transfection in PK-15 cells to determine the effect of PCV3 Cap on the expression level of hnRNP K protein.The results showed that with the increase of transfection time or dose,the expression level of hnRNP K had no significant change(P > 0.05),indicating that PCV3 Cap protein did not affect the expression of endogenous hnRNP K;subsequently,Cap was transfected into PK-15 cells overexpressing hnRNP K.Western blot results showed that the cleavage levels of Caspase-3,-9 and PARP induced by Cap in hnRNP K cell lines were significantly decreased,and the Cyt c released into the cytoplasm was also significantly decreased.These results suggest that PCV3 Cap protein induces mitochondrial apoptosis,and only activates Caspase-dependent mitochondrial apoptosis pathway.hnRNP K protein is the host interaction protein of PCV3 Cap,and plays an important anti-apoptotic role in mitochondrial apoptosis induced by Cap protein.