The Proapoptotic Mechanism Of Porcine Circovirus Type 2 ORF4 Protein

Porcine Circovirus 2(PCV2)was not only the smallest viras in the mammalian cells,but also the main pathogen of post-weaning multisystemic wasting syndrome(PMWS).Previews studies considered the pathogenesis of PCV2 were relevant to various kinds of factors,among them the apoptosis caused by virus played a key role in pathogenic mechanism.ORF4 was once regarded as a viral protein involved in apoptosis,but how it affected apoptosis was still unknown.In this study,we observed the subcellular localization of ORF4 protein,detected the apoptosis induced by ORF4 or ORF4 deficient virus and explored the possible cell proteins which bind to ORF4.All in all,the study was aimed to elucidate the potential function of ORF4 and its mechanism of apoptosis.1.PCV2 ORF4 potential biological propertyThe existence of PCV2 ORF4 was already demonstrated through Northern blot and indirect immunofluorescent assay(IFA).However,ORF4 could not be detected by Western Blot.This study,we found proteasome inhibitor MG 132 can slow down the degradation of ORF4 when ORF4 was transfected in the cells.Moreover,ORF4 was not detected unless MG 132 was added in ORF4-overexpressing cell line.It was concluded that ORF4 was unstable and susceptible to proteasome degradation.This property of ORF4 also exists in PCV2 infected cell,since immunofluorescence positive cells were increased in cells with MG132.Although ORF4 was still not detected in PCV2-infected cells by western blot in this study,these data could be used for future reference in ORF4 subsequent detection.2.Analysis of PCV2 ORF4 subcellular localizationIn the study,the subcellular localization of PCV2 ORF4 was predicted by online software,such as CELLO v.2.5:subCELlular predictor.Then confocal laser scanning microscope was used to observe ORF4 localization in PCV2 infected and Flag-ORF4 transfected PK15 cells.The result showed that ORF4 was completely located in the mitochondria during transfection,while targeted to both mitochondria and nucleus at the late stage of PCV2 infection.Further analysis was performed in ORF4-expressing cell line and found ORF4 almost all diffused distributing in the nucleus.These data suggest ORF4 could have double locations in the cells.Besides,the a-helices and transmembrane hydrophobic region within ORF4 also been predicted and a series of GFP-fusion constructs harboring ORF4 segments which encompass or omit a potential ?-helix were used to measure the possible mitochondrial targeting sequence(MTS)of ORF4.The result showed that,the deletion of amino acids 1 to 30 exerted dotted distribution in the cytoplasm suggesting mitochondrial targeting and indicating ORF4 containing an amphipathic ?-helix at the N terminus of ORF4(amino acid residues 24-30)with preceding 24 basic residues is responsible for the mitochondrial targeting.3.Analyses of PCV2 ORF4 functionTo analyze the function of ORF4 protein.ORF4 was overexpressed in different cell lines including PK15,293T and 3D4/31 to detect the prominent indicators of apoptosis.The results showed that cleavage of caspase-3 were increased in all transiently transfected cells.In contrast,the cleaved casp-3 has no significant alteration in the ORF4 stably expressing PK15 cell line.Study on the apoptosis pathway activated by transiently expressed ORF4 revealed that the cleavage of caspase 9 was increased,a higher level of cyt c release into the cytosol from the mitochondria,but AIF which involved in caspase-independent apoptosis still not detected outside of mitochondria.These results indicated that ORF4 transiently expressed induced apoptosis via caspase-dependent mitochondria pathway.Searching for the differences between two states of ORF4 reveal that the activation of apoptosis by ORF4 is based on its mitochondrial localization.For further confirmation of the role of ORF4 protein in apoptosis during infection,wild type PCV2(rPCV2)and ORF4 mutant PCV2(m4PCV2)were infected PK15 cells for 72h,respectively.The results showed that m4PCV2 induce lower rate of apoptosis in comparation with rPCV2,including lower proportion of TUNEL-positive cells and robust decrease in the level of caspase-9,caspase-3 cleavage and cytc release.Overall the results of transfection and infection confirmed ORF4 played an important role in inducing apoptosis through mitochondria pathway during both ORF4 transfection and PCV2 infection.4.Study on the mechanism of apoptosis induced by PCV2 ORF4To determine the mechanism within ORF4 induced mitochondrial apoptosis.The interaction between ORF4 with VDAC1,ANT3 and COX8A were detected by coimmunoprecipitation and the results showed ORF4 only interacted with ANT3.It was further confirmed by colocalization of ORF4 and ANT3 in the mitochondria through three-dimensional structured illumination microscopy(3D-SIM).To examine whether MTS of ORF4 mediated its interaction,deletion mutants of ORF4 was used in Co-IP and GST-pull down assays.The results showed that the MTS(1-30aa)of ORF4 was responsible for the interaction between ANT3 and ORF4.When the expression of ANT3 in PK15 cells was interfered by specific shRNAs-mediated knockdown,not only the caspase3 cleavage downregulated in ORF4 overexpressed cells,but also the release of cyt c and caspase3,caspase9 cleavages were largely decreased during PCV2 infection.These results suggested that ANT3 play a critical role in ORF4 alone or PCV2-induced mitochondrial apoptosis.