| Tumor suppressor p53 plays an important role in mediating apoptosis and cell cycle capture in response to different stress including virus infection, and regulating host antiviral defense is new function of p53. Influenza A virus (IAV) induces apoptosis of infected cells, p53 is accumulated and activated in IAV infected cells, the results showed biphagic patterns of p53 accumulation and activity, which is associated with IAV replication.This study was undertaken to examine the mechanism of p53 accumulation and activity in IAV-infected cells. Here we show that p53 accumulation in IAV-infected cells resulted from protein stabilization, which was associated with a weakened p53-Mdm2 interaction. In IAV-infected cells, p53 was stabilized and its half-life was remarkably extended. The ladders of poly-ubiquitinated p53 were not detectable in the presence of proteasome inhibitor MG132 and less sensitive to proteasome-mediated degradation during IAV-infection.In the infected cells where Mdm2 is responsible for regulating p53 mono-ubiquitination, neither a lack of abundance nor altered sub-cellular distribution of Mdm2 was observed, furthermore, poly-ubiquitination ligase p300 expressed in a higher level than uninfected cells. However, a weakened p53-Mdm2 interaction was detected by co-immunoprecipitation. In addition, an increase in phosphorylation of p53 at Thr18 and Ser392 was observed. We propose that the mechanism underlying the increased p53 stabilization is that IAV infection induced a weakened p53-Mdm2 interaction, which led to a decrease in ubiquitination levels of p53, consequently resulting in the increased p53 stabilization. The increase in phosphorylation of p53 at Thr18 presumably contributed to the weakened p53-Mdm2 interaction.Viral proteins play important roles in resistance to host anti-viral effects, especially its non-structural protein NS1 have played a variety of important functions during the intracellular signaling pathway. In interferon deficient Vero cells, p53 luciferase activities were detected by co-transfection experiments and dual reporter genes system. The results showed that NS1 inhibits the activity of p53 remarkably. However, NS1 does not affect the p53 protein levels and subcellular localization as detected by western-blotting and indirect immunofluorescence assay. NS1 and p53 were co-transfected into H1299 cells, apoptosis were detected by flow cytometry and apoptosis detection kit TUNNEL. The results showed that NS significantly inhibits p53-mediated apoptosis. To construct different deletion mutants of NS1, modified PCR-based site-directed mutagenesis was performed. To screen domain responsible for inhibiting p53 activity, p53 luciferase activity were detected by dual reporter gene systems. The results showed that the 144-188 amino acid of NS1 protein is a key domain responsible for inhibiting p53 activity. The flag-NS1-â–³144/188 did not inhibit p53 activity. Furthermore, the flag-NS1-â–³144/188 was unable to inhibit p53-mediated apoptosis. H1299 cells were co-transfected with GFP-p53 and Flag-NS1-wt or flag-NS1-â–³144/188 respectively. The results showed that NS1 associates with p53, and flag-NS1-â–³144/188 mutant was also able to bind p53 as detected by Co-IP experiment. So NS1 bound to p53 was one of mechanisms underlying the inhibition of p53 activity, the 144-188 domain of NS1 protein is an inhibitory domain but not a binding domain.In addition, viral protein NP was found to activate p53 activity, but did not activate p53 mediated apoptosis. It regulated the activity of p53 by competing with the NS1. The domain responsible for activating p53 was located at the C-terminal of NP. The detailed mechanism and the biological significance of activation p53 by NP need uncover in the future.Finally,this study analysis the mechanism of dynamics of p53 activity in influenza virus infected cells, the virus proteins involved in regulating p53 activity. This may be one of the mechanisms that influenza viruses escaped host innate immune. Those researches provided theoretical basis for study of anti-influenza virus drug develop- ment and model for influenza virus and host-cells interaction researches in the future.
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