【作者】 王晓杜；

【导师】 马志永；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2010， 博士

【摘要】 肿瘤抑制因子p53在细胞中具有细胞周期捕获、诱导细胞凋亡和抗病毒等功能。流感病毒感染细胞后,诱导被感染细胞发生凋亡,胞内的p53分子蛋白水平和活性呈现双相性上升的变化,这种变化是与病毒的复制密切相关。本研究首先探讨猪流感病毒感染细胞后p53分子活性的变化机制。由于前人研究结果表明p53蛋白水平的增加和活化与其翻译后修饰有关,而与转录水平没有关系。本试验利用Real-time PCR和半定量PCR分别检测病毒感染MDCK细胞p53分子mRNA水平,结果表明猪流感病毒感染并没有使p53转录水平发生改变。利用蛋白质合成抑制剂放线菌酮处理猪流感病毒感染的Vero和MDCK细胞,Western-blotting检测p53分子稳定性的变化,结果表明病毒感染组p53稳定性比未感染组明显上升,证实病毒感染细胞p53蛋白水平的增加是由于其稳定性增加造成的。利用蛋白酶体抑制剂MG132处理流感病毒感染的Vero细胞,结合使用放线菌酮,Western-bloting检测p53分子蛋白水平的变化,结果表明感染组和未感染组在降解途径受到抑制时p53稳定性没有明显差异,所以流感病毒感染使p53分子稳定性增加是与蛋白酶体降解途径密切相关。p53分子需要泛素化标签才能通过蛋白酶体途径进行降解,本试验利用蛋白酶体抑制剂MG132处理流感病毒感染的CV1和Vero细胞,检测泛素化p53的水平,结果表明病毒感染细胞组p53泛素化水平明显比未感染组少,说明流感病毒感染明显减少p53泛素化水平。前人研究表明参与p53泛素化的主要酶是Mdm2分子,本试验利用Mdm2抑制剂处理病毒感染的细胞,Western-bloting检测发现在病毒组和未感染组泛素化p53几乎检测不到,所以说明流感病毒感染引起p53泛素化水平减少是通过Mdm2介导途径来完成的。本试验利用Western-bloting检测猪流感病毒感染后Mdm2分子蛋白水平的变化,间接免疫荧光检测Mdm2亚细胞定位变化,结果表明流感病毒感染的细胞Mdm2蛋白水平没有发生明显变化,且其亚细胞定位也没有发生改变。p53蛋白的降解需要其多泛素化标签,p53多泛素化连接酶是p300,本试验检测了流感病毒感染MDCK细胞后p300的蛋白水平,发现p300的蛋白水平随着病毒的复制而增加,证明多泛素化p53的减少不是由于p300的表达量引起的。Mdm2与p53发生相互绑定是p53泛素化的必要条件,本试验利用H1299细胞转染外源性GFP-p53,感染流感病毒24h后Co-IP检测Mdm2与p53亲和能力,另外在病毒感染CV1细胞20 h后,Co-IP检测Mdm2与p53亲和能力,结果表明流感病毒感染明显减弱了Mdm2与p53之间的相互作用。前人研究表明Thr18的磷酸化是p53和Mdm2相互作用下降的机制之一,本试验检测了流感病毒感染后p53蛋白392位Ser和18位Thr的磷酸化水平,结果表明这两个位点磷酸化都明显升高,推测流感病毒感染造成p53磷酸化水平升高是导致p53和Mdm2相互作用下降的机制之一。流感病毒复制时其病毒蛋白在抵抗宿主抗病毒效应中发挥重要的功能,特别是其非结构蛋白NS1在胞内各种信号通路中都发挥重要的功能。在Vero细胞中共转染流感病毒各基因真核表达质粒和双报告基因质粒,测定p53相对luciferase活性,结果表明NS1能显著地抑制p53的活性,NP能显著激活p53活性。Western-blotting和间接免疫荧光试验结果表明NS1、NP既不影响p53的蛋白水平又不影响p53的亚细胞定位。NS1和p53共转染H1299细胞,流式细胞术和TUNNEL试剂盒检测细胞凋亡,结果表明NS1能显著抑制p53介导的细胞凋亡。利用PCR定点诱变技术构建不同的NS1突变体,不同突变体与p53-luc双报告基因质粒共转染Vero细胞,测定p53相对luciferase活性,结果表明NS1蛋白的144-188位氨基酸是决定NS1抑制p53活性的关键位点,且flag-NS1-△144/188不能抑制p53介导的细胞凋亡。Flag-NS1-wt及flag-NS1-△144/188和GFP-p53共转染H1299细胞,Co-IP实验结果表明NS1能绑定p53,而flag-NS1-△144/188突变体也能绑定p53,所以这种绑定可能是NS1抑制p53活性的机制之一。另外也筛选到能显著激活p53活性的流感病毒蛋白NP,初步筛选到与激活p53活性相关的位点为NP的C端结构域,但是NP与p53介导的细胞凋亡没有关系,NP激活p53的详细机制和生物学意义需要进一步进行研究。总之,本研究解析了流感病毒感染细胞p53活性变化机制,以及病毒蛋白参与p53活性调节的机制。该机制可能是流感病毒逃逸宿主先天性免疫的方法之一,它为以后研究流感病毒抗病毒药物的开发提供理论基础,也为流感病毒与宿主细胞之间相互作用提供研究模型。更多还原

【Abstract】 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-△144/188 did not inhibit p53 activity. Furthermore, the flag-NS1-△144/188 was unable to inhibit p53-mediated apoptosis. H1299 cells were co-transfected with GFP-p53 and Flag-NS1-wt or flag-NS1-△144/188 respectively. The results showed that NS1 associates with p53, and flag-NS1-△144/188 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.更多还原