Analysis Of The Dynamics Of P53 Protein In Swine Influenza Virus Infected Cells

Astumor suppressor,p53 protein plays important role in mediating the cell cycle and promoting the cellular apoptosis.In recent years,researcher discovered that p53 also function in the innate antiviral response.Turpin described that p53 was induced in the influenza virus-infected cells,but the increased p53 only occurred at the apoptotic stage.It was unknown how the p53 protein inhibits the replication of influenza virus.In this paper,we aim to discover the dynamics and biological activity of p53 in the cell infected with the swine influenza virus to explore the possible way that p53 used to inhibit the replication of influenza virus.In order to identify the p53 status in the MDCK and Vero cells,we analyze the expression of p53 and the downstream genes transactivated by p53 such as IRF9 and Bax in the MDCK and Vero treated with 10μM 5-Fu with western blot and indirect immunofluorescent assay.The results showed that the expression of p53,IRF9 and Bax increased in the 5-Fu treated MDCK and Vero.So,the p53 status in MDCK and Vero cells is normal which can be used in the following experiments.To explore the dynamic change of p53 protein in the SIV infected cells,we analyzed the p53 protein in the cells collected at different time post viral infection with western-blot.We also checked the expression of p53 protein in the MDCK infected with heat-inactivated virus and UV-inactivated virus to investigate the relationship of the dynamics of p53 protein and the replication of the SIV.At the same time,the transcription of p53 gene in the SIV infected cells was detected with the semi-quantitative RT-PCR.The results showed that the p53 protein increased in a biphasic method in the SIV infected cells.The first elevation appeared at 1-3h in MDCK or 4-8h post infection,the second increase happened at 10-16h in MDCK or 14-18h in Vero post infection.The expression of p53 was stable in the MDCK infected with heat-inactivated virus,but the first increase of the p53 protein could be discovered in the MDCK infected with UN-inactivated SIV.The transcription of p53 gene in the SIV infected cells didn't change.So,we could draw the conclusion that the up-regulation of p53 protein in the SIV infected cells appeared biphasic which was correlative with the viral replication but independent of the transcription of p53 gene.To verify the transcriptional activity of the increased p53 protein in the SIV infected cells,we investigated the expression of the Bax and IRF9 genes transactivated by the p53 in the SIV infected MDCK cell.We also checked the change of the Bax and IRF9 proteins in the MDCK treated with the 20μMα-pifithrin,a specific inhibitor of p53 activity,after infected with SIV to exclude the possibility that the increased expression of p53 transactivated genes was independent of the p53 transcriptional activity.The results showed that increased p53 could up-regulate the expression of the Bax and IRF9,but not in theα-pifithrin treated cells.So,the increased p53 protein in the SIV infected cells has the transcriptional activity.In order to elucidate the correlation of the increased p53 with SIV replication stage,we administered the viral protein NS1 and the viral titer in the MDCK and Vero cells collected at different time with western-blot and TCID50 differently.The results suggested that the viral NSlinitially appeared at the 10h in Vero and at the 6h in MDCK with little progeny virus between the two p53 increase phases.So,the first p53 increase happened at the early stage of the viral replication and the second p53 elevation appeared at the middle-late stage of the viral replication.In order to explore the ability of the p53 to inhibit the SIV replication,we established the p53 knock down MDCK cell line with RNAi technique.The replication of SIV in p53 knock down MDCK was investigated.The apoptosis and the cell cycle arrest in the MDCK infected with SIV were also checked with flow cytometry.The results showed that the viral title increased in the p53 knock down MDCK,the cell cycle arrest didn't appear at the early phase of viral replication and the apoptosis happened at the middle-later stage of viral replication.As a whole,we first discovered the p53 protein increased in biphasic way in the SIV infected cells.The first p53 protein increase appeared at the early stage of viral replication.The first increased p53didn't induce the cell cycle arrest,but which could transactivated the expression of the IFN regulative factor 9(IRF9).ln this stage,the increased p53 protein maybe inhibit the viral replication through the IFN signal pathway.The second increased p53 happened at the middle-late stage of SIV replication which was correlative with the apoptosis.