【作者】 朱紫祥；

【导师】 马志永；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2013， 博士

【摘要】 肿瘤抑制因子p53被称为“基因组卫士”，在细胞的DNA修复过程中发挥着极为重要的作用。同时p53在细胞中具有诱导细胞凋亡、参与细胞周期捕获、参与细胞代谢和抗病毒等功能。流感病毒感染细胞后，p53的表达量得以累积，IRF9的表达也明显上升。流感病毒感染p53敲除型小鼠与p53野生型小鼠后，p53敲除型小鼠的临床症状、肺脏的病理变化及流感病毒滴度均要严重于p53野生型小鼠。这表明p53与宿主抗流感病毒反应具有着密切的关系。本研究首先利用流感病毒感染p53正常及p53下调表达的A549细胞，对细胞中病毒的复制水平和病毒滴度差异进行比较，了解清楚p53是否直接参与细胞抑制流感病毒复制的过程。同时检测Ⅰ型干扰素信号通路中多个抗病毒基因的表达差异，间接反映流感病毒感染后，p53对Ⅰ型干扰素信号通路的影响。结果表明，流感病毒感染后，p53直接影响着Ⅰ型干扰素信号通路中多个抗病毒相关基因的表达；下调p53表达后，IRF9、IRF7、RIG-I、ISG20、ISG15、GBP1和OAS1等多个基因的mRNA表达量均显著下降。Ⅰ型干扰素通路在机体抗流感过程中发挥着非常重要的作用。这表明流感病毒感染后，p53可以通过影响Ⅰ型干扰素通路而参与宿主抗流感病毒作用。为了进一步验证这种关系并不是单纯由于流感病毒所引起，而是p53与Ⅰ型干扰素通路之间直接的联系。我们利用Ⅰ型干扰素处理p53正常及p53下调表达的细胞，检测了Ⅰ型干扰素信号通路中多个抗病毒基因的表达差异。结果发现了与流感病毒感染后相类似的结果，而且差异程度较流感病毒感染后所表现的更为明显。同时，通过Ⅰ型干扰素对干扰素刺激应答元件(ISRE)的激活实验，我们发现ISRE的激活必须要有p53的存在才能够完全激活，这些结果均表明p53具有直接促进Ⅰ型干扰素通路激活的功能。p53启动子序列中被报道含有Ⅰ型干扰素作用元件，Ⅰ型干扰素可以转录调控p53的表达。而本研究发现p53具有直接促进Ⅰ型干扰素通路激活的功能。这表明p53与Ⅰ型干扰素可以相互促进、相互协调发挥抗病毒作用。Ⅰ型干扰素在机体发挥抗流感过程中发挥着极其重要的作用。GBP1作为干扰素诱导的抗病毒蛋白已被报道可以抑制多种病毒的复制，但关于GBP1是否在流感病毒感染过程中发挥着抗病毒作用至今仍没有报道。最近，有报道发现流感病毒感染后GBP1的表达量显著上调。干扰素诱导的GBP1的表达可以被流感病毒抑制。这些研究结果表明GBP1与流感之间存在着一定的联系。本研究通过hGBP1的过表达实验证明了hGBP1具有抑制流感病毒复制的能力。hGBP1K51A的突变会使得hGBP1丧失其与GTP结合的能力，进而导致GTPase活性的丧失。hGBP1K51A的过表达不能够抑制流感病毒的复制，这表明GTPase活性是hGBP1发挥抑制流感病毒复制所必需的。NS1作为流感病毒拮抗宿主免疫反应的重要分子，可以与多类抗病毒蛋白直接结合，拮抗这些蛋白的抗病毒能力。我们研究发现NS1也可以和hGBP1相结合，NS1效应区123～144位氨基酸组成的片段与hGBP1第51位赖氨酸是两者相结合所必需的关键位点。GTPase是hGBP1发挥抗流感病毒的重要基础。那么NS1是否会抑制hGBP1的GTPase活性？研究发现NS1可以显著抑制hGBP1的GTPase活性，进而拮抗hGBP1介导的抗流感病毒能力。总之，流感病毒感染后，细胞的hGBP1在转录和蛋白水平上均显著上升。过表达hGBP1可以抑制流感病毒的复制。hGBP1第51位赖氨酸是hGBP1发挥抗流感作用所必需的。流感病毒NS1可以与hGBP1相结合，hGBP1第51位赖氨酸和NS1第123-144位氨基酸片段是两者发生结合所不可缺少的。NS1可以抑制hGBP1的GTPase活性，进而拮抗hGBP1发挥的抗病毒作用。hGBP1在机体发挥抗流感病毒免疫过程中具有着重要作用，流感病毒NS1可以显著拮抗这种抗病毒能力。hGBP1被证明具有抑制流感病毒复制的能力。干扰素、IL-β及LPS等均可以诱导hGBP1的表达。p53作为一种转录因子，与干扰素通路具有着密切关系，p53已被报道可以转录调控多个干扰素诱导基因的表达。本实验室前期研究发现，流感病毒感染p53基因敲除小鼠后，GBP1的表达量明显低于p53野生型小鼠中GBP1的表达量。这表明GBP1与p53之间或许存在着一定的联系。通过ChIP on Chip进行高通量分析后，我们发现GBP1启动子序列能够和p53相结合。而进一步研究发现hGBP1的确为p53的靶基因。DNA损伤、病毒感染及细胞因子的刺激均可以通过影响p53而影响到hGBP1的表达。hGBP1启动子序列-4141～-4112和-2440～-2416两个基序为p53的结合反应元件。p53可以通过转录调控hGBP1的表达发挥抗病毒及抗肿瘤效应。该研究为进一步研究p53抗肿瘤和抗病毒的分子机制，以及开发抗病毒、抗肿瘤药物可以提供一定的参考依据。总之，本研究证明了p53与Ⅰ型干扰素通路之间存在着紧密的联系，p53具有直接促进Ⅰ型干扰素通路激活的功能。p53与Ⅰ型干扰素可以相互促进、相互协调发挥抗流感病毒的作用。hGBP1被证明具有抑制流感病毒复制的能力，其为p53下游的靶基因。p53可以通过转录调控hGBP1的表达发挥抗病毒及抗肿瘤效应。这些研究结果，为今后进一步研究宿主的先天性抗流感病毒的反应机制，提高机体抗流感病毒能力提供了药物设计的靶标分子。为控制流感病毒在畜牧业中的危害提供理论基础，也为人类防治流感病毒的蔓延和扩散提供了一定的视角。更多还原

【Abstract】 Tumor suppressor p53is widely known as ‘the guardian of the genome’, that performs importantfunctions in DNA repair process. In addition, p53has also been proven to regulate various biologicalprocesses such as cellular apoptosis, cell cycle arrest, cellular metabolism and antiviral responses.Influenza A virus (IAV) infection can induce the accumulation of p53, which results also in theupregulation of IRF9. The clinical symptoms, pathological responses and the viral loads of p53knockout (p53KO) mice shows much more severe than p53wildtype (p53WT) mice after IAV infection.All these results imply an important role of p53to perform antiviral activity against IAV infection.A549cells with regular and deficient expression levels of p53were infected by IAV respectively. Theviral replication ability and the viral titers of the cells were determined and compared to understand theeffects of p53in IAV infection. Besides, the expression of several antiviral related genes in type Iinterferon (type I IFN) signaling pathway were detected to confirm that p53can affect the activation oftype I IFN signaling pathway during IAV infection. The results indicated that, p53directly affects theexpression of several antiviral related genes in type I IFN signaling pathway. Impaired expression ofantiviral related genes was found in the p53knockdown (p53KD) A549cells during IAV infection, suchas IRF9, IRF7, RIG-I, ISG20, ISG15, GBP1and OAS1. The IFN signaling pathway plays a key role inregulation of immune response against IAV infection，the results suggested that p53performs anti-IAVability through regulating the activation of type I IFN signaling pathway. To further prove the regulationof p53to type I IFN signaling pathway is an universal response, but not just in IAV infection cells. A549cells with regular and deficient expression levels of p53were treated with type I IFN, the expression ofseveral antiviral related genes in type I IFN signaling pathway were detected. In response to type I IFN,the impaired expression of antiviral related genes was also found in the p53KD cells, the impairedextent was more notable than the IAV infection groups. In addition, p53was found to be essential forthe activation of IFN-stimulated response elements (ISREs) induced by type I IFN. These resultsindicated p53can reinforce type I IFN signaling pathway. The promoter sequence of p53contains ISREfor type I IFN, so type I IFN can upregulate p53expression. Our results demonstrated p53can reinforcetype I IFN signaling pathway. It suggests a crosstalk between the p53and type I IFN pathway, p53andtype I IFN cooperates with each other to defend the cells from virus infection.Type I IFN is indispensable for host to perform antiviral effects against IAV. GBP1has beenreported to possess the antiviral activity against several viruses. But whether IAV is inhibited by GBP1is still not clear. It is reported that, IAV infection induces GBP1expression, IAV inhibits the productionof GBP1induced by IFN. It suggests that GBP1may be involved in antiviral responses during IAVinfection. Overexpression of hGBP1was found to inhibit IAV replication in this study. The K51locatedin the highly conserved phosphate-binding loop of hGBP1is critical for its biological activity andfunction. Mutation of lysine51to alanine (K51A) abrogates GTP binding, dimerization, and GTPhydrolysis. Overexpression of hGBP1K51A failed to inhibit IAV replication, which suggests the GTPase of hGBP1is essential for its anti-IAV activity. NS1is widely known as an antagonist of hostimmune response to facilitate IAV replication. NS1was reported to interact with various anti-IAVproteins to antagonize their antiviral activity. NS1was also found to interact directly with hGBP1. Theresidues of123-144in the effector domain of NS1were required for the binding. The K51of hGBP1also decided the interaction. GTPase was proved to be essential for hGBP1to inhibit IAV replication;we predicted that NS1binds with hGBP1perhaps affects its GTPase activity. The ELIPA assay wascarried out to confirm this deduction, which showed the GTPase activity of hGBP1was significantlyimpaired by NS1. NS1clearly antagonized the anti-IAV activity of hGBP1. In conclusion, IAV infectiontriggers hGBP1expression. hGBP1possesses the anti-IAV activity, the GTPase of hGBP1is essentialfor its anti-IAV activity; the GTPase is dependent on the51K residue. NS1of IAV interacts with hGBP1,the51K of hGBP1and the residues of123-144of NS1are required for the interaction. NS1inhibits theGTPase of hGBP1to antagonize the anti-IAV activity of hGBP1. hGBP1shows an important effect inthe anti-IAV responses, NS1can weaken hGBP1-mediated anti-IAV ability.Now, hGBP1is proved to possess anti-IAV activity. hGBP1expression can be induced by IFN, IL-βand LPS. p53, as a transcription factor shows important effect on IFN signaling pathway. Many ISGshave been proved to be p53transcriptional target genes. Our previous study showed that IAV infectioninduced GBP1expression was significantly impaired in p53KO mice compared with p53WT mice;which implied p53may correlate with GBP1expression. ChIP on Chip assay was carried out to identifythe potential transcriptional target genes of p53. The promoter sequence of GBP1was found to bindwith p53, and GBP1was identified as a potential target gene. We further confirmed that hGBP1is adirect transcriptional target gene of p53. DNA damage, viral infection and cytokines triggeredexpression of hGBP1was clearly regulated by p53. The fragments of-4141/-4112and-2440/-2416athGBP1promoter sequence were confirmed as the p53response elements. All these results indicated p53may transactivate hGBP1gene to perform the antiviral and antitumor activity. In conclusion, theseresults provided a new insight into antiviral and antitumor therapy and paved the way for future researchin p53functions.In briefly, this study proved the correlation between p53and type I IFN pathway. p53possesses theability of enforcing type I IFN pathway activation. p53cooperates with type I IFN to perform anti-IAVresponses. hGBP1gene is a target gene of p53, p53upregulates hGBP1expression to carry out theantiviral and antitumor effects. Overall, all the results in this study provided a theoretical basis for hostanti-IAV immune responses research and anti-IAV strategies application to prevent the threat, damageand economic loss caused by IAV infection; which also offered several target molecules for anti-IAVmedicines development.更多还原