Signal Transduction Pathways Of TGEV-induced Apoptosis In PK-15Cells

Transmissible gastroenteritis virus (TGEV) belongs to the family of Coronaviridae,subfamily coronavirinae, genera alpha-coronavirus. TGEV commonly replicates in thedifferentiated enterocytes of the small intestine and accordingly, causes similar clinicalsymptoms with lethal watery diarrhea and dehydration in piglets. TGEV infection in ST cellsand PK-15cells generally induces (cytopathic effect) CPE and eventually causes cell death.But the molecular signal transduction pathways caused by CPE are not clear. In this study, weused TGEV to infect PK-15cells. First, the change of cell cycle was detected to determinecell cycle arrest. Then apoptosis related indexes were detected to determine cell apoptosisoccurrence. Last, the apoptotic signaling pathways induced by TGEV and N protein werestudied scientificly. The results were as follows.1. Flow cytometry was used to determine the effect of TGEV infection on cell cycleprofiles. Results showed that there was a significant increase in the proportion of cells in theS and G2/M phases of the cell cycle in TGEV-infected asynchronous and quiescent PK-15cells. Western blot analysis showed that TGEV infection increased the protein level of p53and resulted in phosphorylation of p53at serine15and20, and increased the level of p21,decreased cdc2, cdk2and cyclin B1, suggesting that TGEV could mediate cyclin and cdks toinduce cell cycle arrest. PFT-α (p53inhibitor) treatment attenuated TGEV-induced the arrestof the cell cycle, prevented p21expression, increased cyclin B1, cdc2and cdk2protein levelsin TGEV-infected cells. These results suggest that p53might play a key role in TGEV-induced cell cycle arrest. UV-inactivated TGEV lost the ability to induce cell cycle arrest. Inquiescent PK-15cells, the virus replication volume was significantly lower than inasynchronous cells, while in S and G2/M phase synchronized cells, the virus replication wassignificantly higher than in asynchronous cells, suggesting that cell cycle arrest in S andG2/M phase might benefit to virus replication.2. Fluorescent microscope observation showed that TGEV-infected PK-15cells hadtypical apoptotic features including chromatin condensation and nuclear fragmentation afterAO/EB staining. Transmission and scanning electron microscope observation showed cell shrinkage, volume reduction, and microvilli disappearance, cell blebbing and membraneembedded apoptotic bodies. The chromosomal DNA fragmentation analysis showed DNAladders could be detected as early as24h p.i., when PK-15cells were infected with TGEV at1MOI, and the intensity of the ladder bands increased with time and dose of infection. Flowcytometry analysis of apoptosis with dual Annexin V-PI cell labeling showed that the numberof apoptotic cells increased over time to gradually move into late apoptotic. These resultsdemonstrate that TGEV infection induces apoptosis in PK-15cells.3. Caspase activity assay and Western blot analysis showed that TGEV infectionactivated caspase-8,-9and-3. Activated caspase-8could cleave Bid to truncated Bid (tBid).PARP, substrate of caspase-3, also degraded. Real-time PCR and Western blot analysisshowed that TGEV infection upregulated the protein levels of FasL and Bax, downregulatedBcl-2, induced Bax and tBid translocation to mitochondria, cytochrome c release to cytosol inTGEV-infected cells. These results suggested that TGEV can activate FasL-mediated deathreceptor pathway and mitochondrial-mediated pathway to induce apoptosis in PK-15cells.NH4Cl treatment not only reduced viral progeny production in a dose-dependent manner, butalso attenuated TGEV-induced apoptosis, suggesting that TGEV induction of apoptosisrequires the viral particles to undergo uncoating. UV-inactivated TGEV lost the ability ofapoptosis induction, suggesting that TGEV induction of apoptosis require virus replication.4. Western blot analysis showed that TGEV infection resulted p38MAPKphosphorylation, p300/CBP, MDM2decrease, p53accumulation and phosphorylation atserine15,20and46, transcriptional activity of p53enhancement. PFT-α (p53inhibitor) coulddown-regulated FasL and Bax, whereas up-regulated Bcl-2in TGEV-infected PK-15cells.SB203580(p38MAPK inhibitor) treatment attenuated phosphorylation of p53and reducedp53-transcriptional activity, increased the protein levels of p300/CBP, MDM2inTGEV-infected cells. Moreover, TGEV infection significantly increased ROS levels. TheROS scavenger NAC and PDTC treatment significantly reduced phosphorylation of p38MAPK and p53. These results showed that the upstream pathways of death receptor pathwayand mitochondrial-mediated pathway, ROS, p38MAPK and p53signalings participatedTGEV-induced apoptosis. Furthermore, UV-inactivated TGEV lost their ability to increaseROS production, to induce p53and p38MAPK phosphorylation and activate p53transcriptional activation. PFT-α, PDTC, NAC and SB203580inhibited the titer of TGEV,respectively, suggesting that ROS, SB203580and p53might play important roles in thereplication of TGEV in PK-15cells.5. Flow cytometry and Western blot analysis showed that TGEV N protein increased p53and p21expression and regulated cyclin A-cdk2and cyclin B1-cdc2to make cell cycle arrest in S phase and G2/M phase. In addition, TGEV N protein promoted Bax translocated tomitochondria and cytochrome c released to cytoplasm, activated caspase-3, to induce cellapoptosis.This study illustrates the molecular mechanism of TGEV-induced apoptosis and theregulation of TGEV N protein in apoptosis. The results provide theoretical basis for thepathogenesis of TGEV and new ideas for exploiting target drug to resist TGEV.