Mechanisms Of Mitochondrial Apoptosis And Mitophagy In Porcine Circovirus Type 2 Infected Cells

Porcine circovirus(PCV)is a small,nonenveloped,circular single-stranded DNA virus belonging to the genus Circovirus of the Circoviridae family.PCV2 infection is immunosuppressive,as shown by lymphoid depletion in lymph nodes of infected pigs,and poses a severe threat to the pig industry worldwide.The virus has a circular single-stranded DNA genome of about 1.7 kb.It contains three major open reading frames(ORFs);ORF1 codes for the replicase(Rep)protein involved in viral replication,ORF2 for the capsid(Cap)protein,and ORF3 for a putative protein with proapoptotic activity.The role of proapoptotic ORF3 in PCV2 pathogenesis remains controversial.Our previous studies demonstrated that porcine circovirus type 2(PCV2)triggers an unfolded protein response(UPR)by activating the protein kinase R-like endoplasmic reticulum kinase(PERK)/eukaryotic initiation factor 2?(eIF2?)pathway of endoplasmic reticulum stress(ERS),which in turn facilitates viral replication.PCV2 infection upregulated calcium/calmodulin-dependent protein kinase kinase-beta(CaMKK?)by increasing cytosolic Ca2+ via inositol 1,4,5-trisphosphate receptor(IP3R)to activate autophagy subsequently.In this study,we attempted to address:(1)whether PCV2 could cause mitophagy and the link between mitochondrial apoptosis and mitophagy.(5)The relationship between mitochondrial apoptosis and UPR and the role of Ca2+ and ROS in mitochondrial apoptosis;1.Mechanism of mitophagy induced by PCV2 infection1)PCV2 induced mitophagy in PK-15 cellsTo examine whether PCV2 could induce mitophagy,PK-15/EGFP-LC3 cells were infected with PCV2 for confocal microscopy analysis of mitophagosome formation by merging GFP-LC3 puncta with MitoTracker Red.It shows that the mitophagic vacuoles and lysosomes were colocalized in PCV2-infected PK-15 cells.Fluorescent intensity was higher in PCV2-infected cells than mock-infected ones(p<0.01).TEM images revealed mitochondrial swelling and mitochondria lacking cristae in PCV2-infected cells.PCV2 infection induced autophagic vacuoles,suggesting occurrence of autophagy of damaged mitochondria.These data suggest that PCV2 infection induced mitochondrial damage and might induce selected elimination of damaged mitochondria by autophagy.2)PCV2 induced oxidative stress,activated Drpl and PINK1/Parkin pathwaysTo explore whether PCV2 induces Drpl translocation to mitochondria,PCV2-infected cells were analyzed by confocal microscopy using a specific antibody that recognize the Ser616-phosphorylated Drpl(p-Drp1).We found significant elevation of p-Drp1 intensity in PCV2-infected cells with its translocation to mitochondria.Western blotting and flow cytometry showed that there was significant increase of both p-Drp1 and ROS with the progression of PCV2 infection.NAC treatment was effective in suppressing ROS generation in PCV2-infected cells and in inhibiting Drpl phosphorylation,suggesting that ROS is involved in Drp1 phosphorylation.And it was revealed that NAC significantly reduced mitophagosome formation in PCV2 infected cells(p<0.01).We observed that PCV2 infection led not only to autophagy shown as marked increase of the LC3-?/LC3-? ratio,but also to elevated expression of PINK1 and Parkin in PK-15 cells as well as increased Drpl phosphorylation,indicative of mitophagic responses.Further analysis revealed that increased Parkin accumulation in the mitochondrial fraction of PCV2 infected cells could be blocked by NAC treatment(p<0.05).Thus,we provide clear evidence that there is causal link between mitophagy and excessive ROS production during PCV2 infection,and that ROS might function as an upstream signal for activation of Drpl phosphorylation with subsequent mitochondrial fission and mitophagy.3)Suppression of mitochondrial fission protein Drp1 inhibited PCV2-induced mitophagy and mitochondrial apoptosisTo gain insight into the role of Drpl in PCV2-induced mitophagy and mitochondrial apoptosis,we tested whether specific suppression of Drpl with lentivirus-mediated shRNA(shDrp1)or chemical treatment with Mdivi-1 would affect mitophagic and apoptotic responses.Chemical inhibition of Drp1 or RNA silencing with shDrp1 could effectively alleviate mitochondrial fragmentation caused by PCV2 infection.We found that inhibition of Drp1 expression resulted in decreased PINK1 and caspase-3 cleavage as well as diminished Parkin accumulation and reduced LC3?/? ratio in the mitochondrial fraction(p<0.05 as compared with those of the cells infected with PCV2 only).Down-regulation of Drp1 by Mdivi-1 treatment or shDrp1 significantly decreased ROS and rescued MMP in PCV2-infected cells(p<0.05).Drp1 suppression inhibited loss of mitochondrial mass in cells infected by PCV2(p<0.05,p<0.01 vs control cells).Treatment either with Mdivi-1 or shDrpl could alleviate apoptotic responses in PK-15 cells,shown as decreased apoptotic rate(p<0.05)and reduced activities of caspases-3 and-9 that are closely related to mitochondrial apoptosis(p<0.05).These findings indicate that Drp1 might function as an upstream molecule and cooperate with ROS to regulate the PINK1/Parkin pathway as well as mitophagic response.2.Mechanism of mitochondrial apoptosis in PCV21)Different PCV2 strains induced ER stress,UPR,and apoptosisTo determine whether different strains of PCV2b and PCV2d could induce the UPR and apoptosis,FCM and TUNEL assay were used to detect levels of apoptosis.Western blotting was employed to detect the expression of targeted molecules in PCV2-infected cells.The expression of glucose-regulated protein 78(GRP78),phosphorylated PERK(p-PERK),and phosphorylated eIF2?(p-eIF2?)was significantly increased in virus-infected cells,suggesting that virus infection induced ER stress and the UPR(p<0.05).The viruses also induced apoptotic responses of PK-15 cells,as shown by significant elevation of cleaved caspase-3 levels and increased numbers of apoptotic cells(p<0.05).2)PCV2 induces ORF3-independent mitochondrial apoptosis via PERK activation and elevation of cytosolic calciumThe PCV2b strain YW was used to construct an isogenic mutant through inactivation of the start codon of orf3.The ORF3-deficient virus could still induce PERK/eIF2? activation,caspase-3 cleavage,and GRP78 expression and apoptosis in both PK-15 and PAM cells,although the responses were generally lower than those observed with the parental strain(p<0.05).These results suggested that PCV2 infection induced an apoptotic response independent of the ORF3 protein,probably via the UPR pathway.We found that infection with the ^ ORF3 mutant or its parental strain could raise both cytosolic and mitochondrial Ca2+ levels significantly,while the parental virus showed more pronounced effects(p<0.05).Infection with either ?ORF3 or its parental strain also resulted in more pronounced reduction of MMP with a longer period of infection.Flow cytometric analysis revealed that cells infected with both viruses had higher levels of cytosolic ROS than the uninfected cells,although the impact was more apparent with the parental virus infection.All of these results suggested that infection with either ?ORF3 or its parental PCV2 strain could induce ER stress and elevation of cytosolic Ca2+ levels that would be inevitably linked with mitochondrial Ca2+ overload,reduced MMP,and increased ROS generation.To examine whether the ORF3-deficient PCV2 induced mitochondrial apoptosis via ER stress and UPR activation,GSK was used to block the UPR induced by virus infection and a small-molecule chemical chaperone(4-PBA),to relieve ER stress.Both chemicals could alleviate ?ORF3-induced apoptotic responses in PK-15 and PAM cells,as shown by reduced cleavage of caspase-3 and by Annexin V-fluorescein isothiocyanate(FITC)/propidiumiodide(PI)and TUNEL assays.These findings indicated that ?ORF3-induced apoptosis is more likely the result of UPR activation during viral infection.Inhibition of the UPR or ER stress by the two chemicals could ameliorate virus-induced elevation of ROS levels,similar to the effect of NAC.Such treatments also alleviated MMP reduction and led to diminution of cytosolic and mitochondrial Ca2+ levels.Therefore,we suggest that ?ORF3-induced apoptosis might occur via mitochondrial alterations of Ca2+ levels,MMP,and ROS levels as a result of ERS and UPR activation.3)PCV2 induced UPR and mitochondrial apoptosis via Cap expression.To determine whether PCV2 Cap could induce apoptosis via the mitochondrial pathway,cytosolic Ca2+ and ROS levels were analyzed by flow cytometry at 48 h in PK-15 cells transfected with pCMV-Cap or infected with ?ORF3.Cap expression increased cytosolic Ca2+ and ROS levels and reduced the MMP.We found that activities of caspase-3 and-9 were significantly higher in ?ORF3-infected cells and cells transfected with pCMV-Cap than in control cells(p<0.01).By flow cytometric analysis,we found that Cap expression induced significant cell apoptosis.Clearly,Cap-induced mitochondrial apoptosis involved Ca2+,ROS,and perturbed MMP.We silenced PERK via lentivirus-mediated short hairpin RNA(shRNA)transfer to PK-15 cells,which were then transfected with pCMV-Cap or infected with ?ORF3.Western blotting showed that downregulation of total PERK(t-PERK)expression was significant(p<0.01).The downregulation of PERK could counteract the elevation of Ca2+ and ROS levels induced not only by ?ORF3 but also by Cap expression.Loss of MMP,activation of caspase-3 and-9,and apoptosis rates in Cap-expressing or?ORF3-infected cells were also inhibited by PERK knockdown.Thus,we propose that PCV2 might deploy its Cap to induce ER stress,which would ultimately lead to mitochondrial apoptosis by triggering the proapoptotic molecules Ca2+ and ROS.In summary,this study clarified that the mitochondrial homeostasis disbalance caused by PCV2 is accompanied by the occurrence of mitophagy,while PCV2 specifically activates the PINK 1/Parkin pathway,which is regulated by ROS and related to the mitochondrial fission protein Drp1.Inhibiting the phosphorylation of Drp1 alleviates the occurrence of mitophagy and mitochondrial apoptosis caused by PCV2 infection.Different strains of PCV2 could induce UPR and ERS in PK-15 and PAM cells,indicating the universality of this phenomenon.The apoptosis induced by PCV2 is not entirely dependent on ORF3 protein,but the occurrence of mitochondrial apoptosis caused by ROS accumulation and Ca2+ homeostasis disbalance,which can be alleviated by PERK inhibitors and ERS alleviators,indicating that UPR activation plays a regulatory role in the upstream of mitochondrial apoptosis.The experimental results lay a foundation for further exploration of the pathogenesis and clinical diagnosis of PCV2.