The Role Of P53in Type Ⅰ IFN-induced Antiviral Responses Against Influenza A Virus

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.