| Porcine transmissible gastroenteritis (TGE), caused by transmissible gastroenteritis virusof swine (TGEV), is characterized by high mortality and severely retarded growth in pigletsthat dramatically damages the porcine industry. TGEV infection in cultured cells such as STcells and PK-15cells can cause typical cytopathic effect (CPE). But molecular mechanismsof this CPE are not clear. In this study, we aimed to study the effect of TGEV infection on cellcycle in ST cells. Cell cycle profiles and expression levels of proteins involved in cell cyclewere detected to determined molecular mechanisms of cell cycle arrest. Finally, the impact ofTGEV-induced cell cycle arrest on virus replication was investigated. The results were asfollows:1. Determination of cell cycle profiles in TGEV-infected ST cellsThe results of flow cytometry showed that there was a significant increase in theproportion of S and G2/M phases in TGEV-infected unsynchronized ST cells compared tomock-infected cells from6h and18h post infection (p.i.), respectively. Cells were thensynchronized at G0/G1, G1/S and G2/M phase by serum starvation, thymidine andnocodazole, respectively. Those treated cells were infected or mock-infected with TGEV andcell cycle profiles were determined. Results showed that comparing to mock-infected ST cells,TGEV-infected synchronized cells showed cell cycle arrest at S and/or G2/M phases.2. Detection of cell cycle-regulatory proteinsWestern blot was used to detect the expression levels of cell cycle-regulatory proteins.Results showed that there was a down-regulation of cyclinB1, Cdk1, Cdk2and PCNA inTGEV-infected ST cells; whereas Cdk inhibitor p21and p53was up-regulated.3. Role of p53in TGEV-induced cell cycle arrestIn order to investigate the role of p53in TGEV-induced cell cycle arrest, ST cells weretreated with specific p53inhibitor PFT-α prior to infection with TGEV. Results of flowcytometry indicated that PFT-α-treated-TGEV-infected ST cells showed no phenomenon ofcell cycle arrest. Western blot showed that PFT-α treatment attenuated TGEV-induced cell cycle arrest by increasing protein levels of cyclinB1, Cdk1, Cdk2and PCNA, prevention ofp21expression in TGEV-infected cells, which demonstrated that p53might play a key role inTGEV-induced cell cycle arrest.4. The relationship between cell cycle arrest and virus replicationTGEV was inactivated by UV irradiation prior to infection with ST cells, and cell cycleprofiles were determined by flow cytometry. As results illustrated, UV-inactivated TGEV wasincapable of inducing cell cycle perturbations. UV-inactivated-TGEV-infected ST cellsshowed no significant difference in cell cycle profiles, when compared to mock-infected cells,but showed obvious difference comparing to TGEV-infected ST cells. Above conclusionsindicated that cell cycle arrest is dependent on effective viral replication.To investigate whether TGEV-induced cell cycle arrest was favorable for virusreplication, cells are synchronized at different phases and unsynchronously replicating cellsare used as control. RT-PCR was finally utilized to determine the expression level of TGEVN gene in treated cells. Results showed that in quiescent ST cells, N gene expression levelwas significantly lower than in unsynchronously replicating cells, while in G1/S and G2/Mphase synchronized cells, the virus replication volume was much higher than that inasynchronous cells, suggesting that cell cycle arrested in S and G2/M phases might benefit tovirus replication.Briefly, this thesis demonstrated the molecular mechanism of TGEV-induced cell cyclearrest of ST cells at S and G2/M phases, mainly by up-regulating p53and p21,down-regulation of Cdk1, Cdk2and cyclinB1. Besides, ST cells arrested at S and G2/Mphases by TGEV infection might be favorable for viral replication. |
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