The Role Of P53 In Japanese Encephalitis Virus Replication And Pathogenesis

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, induces inflammation of human brain, which is named endemic B encephalitis (JE) in China and causes dysfunction of human central neuronal system. Two main strategies to control JE are vaccine prevention and mosquito eradication, but over 50 000 cases of JE annually with a fatality in symptomatic cases of 30% demonstrates that development of effective antiviral strategies is a necessary supplement to prevent this disease. Elucidation of virus-host interactions could contribute to the development of an effective antiviral strategy. p53, a transcriptional factor, possesses multifunction of tumor suppressor, DNA repair, pro-apoptosis and antiviral activities. Distinctive strategies had been evolved by viruses to overcome or utilize the biological function of p53. However, to date, there is no report about the interaction of p53 and JEV. To investigate the role of p53 in JEV infection, these studies as follows were performed:1. The level of p53 protein and its transcriptional activity were decreased in JEV-infected cells. To investigate the level of p53 protein and its intracellular localization, the A549 and PIEC cells were infected with JEV (multiple of infection [MOI] =5). The immunofluorescence assay (IFA) showed the nuclear accumulation of p53 was reduced in JEV-infected cells. And this reduction is an extensive phenomenon. The western blot (WB) result indicated the level of p53 protein was decreased at the 48h post-infection in A549 cells or at the 12h post-infection in PIEC cells. The nuclear and cytoplasm of JEV-infected cells were extracted and the level of p53 protein was investigated respectively. The result showed the p53 protein both in nuclear and cytoplasm was reduced. The luciferase reporter assay showed in the JEV-infected cells, the transcriptional activity of p53 was remarkably decreased (p<0.05) and the expression of IRF9 was impaired by WB. In addition, the IFA showed that NS3 co-localized with p53 in JEV-infected cell and their interaction was subsequently demonstrated by co-immunoprecipitation (co-IP). In conclusion, in this study, we first found the level of p53 protein and its transcriptional activity were reduced by JEV infection. And the interaction of NS3 and p53 was identified, which suggested NS3 may involve the reduction of p53 protein in JEV-infected cells.2. p53 inhibits the replication of JEV. To investigate the role of p53 in JEV replication, the replication of JEV in p53 knockdown cells was determined. The results of WB and quantity PCR (qPCR) showed that the expression of p53 in A549 cells was effectively interfered by transfecting the p53 siRNA with final concentration of 150nM. And the growth characteristics of p53 knockdown cells weren't significantly difference to control cells. The IFA showed the replication of JEV enhanced in p53 knockdown cells. The level of NS3 mRNA in p53 knockdown cells was higher than that of in control cells by qPCR (p<0.01). WB results also showed the level of NS3 and E protein in p53 knockdown cells were higher than those of control cells. Plaque assay suggested the virus titer in cell culture supernatant of p53 knockdown was 10-100 folds higher than that of control group. Together, we conclude that p53 inhibits the replication of JEV in cell model. 3. JEV induced apoptosis is a p53 independent manner. To investigate the role of p53 in apoptosis induced by JEV infection, the p53 knockdown A549 cells were infected and the apoptotic cell numbers was determined. First, the trypan blue exclusion assay showed the number of dead cell induced by JEV infection in p53 knockdown cells was significantly more than that of control group (p<0.05). The results of Terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling assay (TUNEL) and fluorescence-activated cell sorting (FACS) indicated the ratio of apoptotic cells in JEV-infected p53 knockdown cells was significantly higher than that of control group (p<0.05). According to the result of mouse cDNA microarray (see the chapter 6), the relative expression of pro-apoptotic gene Xaf1 was 89 folds in p53 knockout (p53KO) mice brain than that of p53 wild type (p53WT). The result of qPCR showed the relative expression of Xaf1 in JEV-infected p53 knockdown cells was significantly higher than that of control group (p<0.05). These results indicate that the apoptosis induced by JEV doesn't depend on p53 transcriptional activity; on the contrary, the deletion of p53 promoted the apoptosis induced by JEV.4. p53 mitigates the pathogenesis of JEV in mouse. To investigate the role of p53 in JEV pathogenesis, 4-weeks p53WT and p53KO mice (n=10) were subcutaneously infected with JEV (104PFU). The challenge experiment results showed that p53KO mice appear an earlier occurred time and a shorter course of the disease symptom. The mortality of p53KO mice infected with JEV was higher than that of p53WT mice. The level of NS3 mRNA in peripheral blood determined by real time PCR revealed the viremia of p53KO mice was higher than that of p53WT mice (p<0.05). The immunohistological investigation displayed the cells with positive NS3 antigen staining of p53KO mice brain were more extensive than those of control group. These results indicated the p53 can inhibit JEV replication in mouse model. The pathological observation showed the glial cells in cerebral cortex and hippocampus of p53KO mice brain produced massive inflammatory exudates. The level of TNF-αand IL-6 protein in p53KO mice brain was higher than that of control by ELISA determination (p<0.05). These results revealed that JEV induces a more serious inflammation in brain of p53KO mice.To investigate the host immune response to JEV infection, the ratio of dendritic cells (DC), macrophage (Mφ), CD4 and CD8 T cell in peripheral blood and spleen were determined by FACS. The results showed that: in peripheral blood of uninfected mice, the ratio of DC had no apparently difference between p53KO and p53WT mice, while the ratio of Mφin p53KO mice were remarkably lower than that of p53WT mice; after 4-days JEV infection, the ratio of DC and Mφof p53KO mice was significantly higher than that of p53WT mice. These indicated the propagation of DC and Mφwere regulated by p53 and suggested it may inhibit the related inflammatory factors production. In addition, the FACS results showed the ratio of CD4 T cell in spleen of JEV-infected p53WT mice had apparently elevated, while the propagation of CD4 T cell in spleen of p53KO mice was failed. These results indicated p53 regulated the propagation of CD4 T cell and suggested p53 may integrate into the host immune response by modulating CD4 T cell propagation.5. Differential transcriptomic profile of mouse brain infected with JEV. To elucidate the mechanism of p53 inhibiting JEV and to screen out the gene (s) that potential regulated by p53, the transcriptomic profiles of p53WT and p53KO mice brain were compared. The results showed the mRNA of some proinflmmatory cytokines such as chemokines, interleukins and tumor necrosis factors were up-regulated in p53KO brain compared with those of p53WT brain; the mRNA level of several immune related molecules were down-regulated in p53KO brain; several apoptosis related molecules had a differential expression. Intriguing, the results of quantity PCR and western blot showed the expression of IFITM3 was impaired in p53knockdown cells. Overexpression of p53 promoted the expression of IFITM3 in p53KO MEF. A potential response element of p53 was searched out in sequence of IFITM3 promoter and a luciferase reporter plasmid was constructed. The luciferase activity of IFITM3 promoter was induced by overexpression of Flag tagged p53 and 5-Fu treatment. We concluded that the IFITM3, an IFN inducible gene which possess an antiviral function against JEV, is a potential gene regulated by p53.Conclusion, we had performed a multi-level analysis on the interaction of p53 and JEV. The innate immunity of p53 against JEV infection had been demonstrated and some potential p53 regulated cellular proteins and immune cells related JEV infection had been identified. These studies further revealed the innate antiviral role of p53 and provided valuable information for further study of p53 anti-JEV mechanism and the development of anti-JEV strategies.