The Biological Consequence Of Nucleoprotein (NP) Of Influenza Virus In Activating P53and Roles Of P53in Anti-influenza Virus

Influenza A Viruses, IAV, are enveloped, negative stranded RNA viruses in the familyOrthomyxoviridae. IAV have caused a large variety of diseases in human history, which have caused a lotof lost to both economic development and human health.Infected cells will block the replication of IAV bysome mechanisms, such as the activation of chemotactic, anti-virus cytokine and inflammatory factors. P53as a tumor suppressor and transcript factor, plays an important role in DNA repairing, cell cycle arrest andapoptosis in response to cell separation, aging, DNA damaging, hypoxia, et al. Recently, it is reported thatp53also play roles in anti-viruses, and our group have found that the activity of p53has increased after theinfection of IAV and NS1protein of IAV can inhibit p53. We have do some research about the roles of p53in anti-influenza viruses.1.The biological consequences of NP in activatin p53. We found that NP could active p53byp53-Luciferase reportor assay. We also found that NP and p53had the same sub-celluar location in A549and Vero cells by IFA, which suggest that the two proteins may interact with each other and the suggestionwas confirmed by Co-ip assay and BiFC. In further experiment, we detected the cell cycle and apoptosis ofA549after the transfection of pFlag-NP plasmid and found that NP may play role in cell cycle arrest butnot in apoptosis, which may be a possible mechanism of p53in anti-influenza viruses.2.p53mitigates the pathogenesis of IAV in mouse and p53inhibits the replication of IAV. In thisresearch, eight weeks old p53wild type (p53+/+) and p53knockout (p53–/–) C57BL/6mice wereintranasally inoculated with a sublethal dose of influenza PR8virus (A/Puerto Rico/8/34(H1N1)).IAV-infected p53–/–mice displayed serious clinical signs compared with IAV-infected p53+/+mice. Thesignificant difference of weight loss was observed between IAV-infected p53+/+and p53–/–mice from day3to7post-infection. By day16post-infection,63.6%of IAV-infected p53–/–mice succumbed to IAVinfection, whereas18.2%of IAV-infected p53+/+mice died (p=0.0222). This observation indicated thatp53-deficient mice displayed increased mortality after IAV infection and that p53have an antiviral effectagainst IAV infection in vivo.Tissue specimens including lungs, hearts, livers, kidneys, spleens and brains of IAV-infected micewere collected on day3and6post-infection for pathological analysis. Gross lesions were observed only inlungs among the tissues specimens analyzed. The affected areas of lungs, which show edematous anddusky red in color, expanded gradually from day3to6post-infection. The distribution area of lung lesionsobserved in IAV-infected p53–/–mice was obviously larger than that in IAV-infected p53+/+mice both onday3and6post-infection, suggesting more severe pathological lesions developed in lungs of IAV-infectedp53–/–mice. The lungs from IAV-infected mice were further examined micropathologically, and the p53–/–mice showed more severe alveolar collapse, edema and hemorrhage compared to p53+/+mice. Takentogether, these data suggested that more severe pathological lesions were developed in lungs in the absenceof p53.Viral loads in lungs of IAV-infected p53+/+and p53–/–mice were analyzed on day3and6 post-infection by a virus titration and a quantitative real-time PCR assay. The virus titers present in lunghomogenates from IAV-infected p53–/–mice were significantly higher than that in lungs of IAV-infectedp53+/+mice on day3post-infection, whereas no significant difference was found between IAV-infectedp53+/+and p53–/–mice on day6post-infection. These results were further confirmed by a quantitativereal-time PCR assay.3.Analysis of lymphocytes in spleen. To investigate the immune respone to IAV infection, the ratio ofCD4+and CD8+cell in spleen were accounted by FACS.Lymphocytes were acquired from the spleen ofp53+/+and p53-/-mice on day3post infection of IAV and we found that the numbers of both CD4+andCD8+T cells increased significantly in IAV-infected p53+/+mice compared to that in mock-infected mice,whereas no significant difference in the numbers of CD4+and CD8+T cells was found between IAV-andmock-infected p53–/–mice. The abolished proliferation of CD4+and CD8+T cells in IAV-infected p53–/–mice implied that the immune response for clearance of IAV infection is diminished in the absence of p53.4.Differential transcriptomic profile of mouse lung infected with IAV. To elucidate the mechanism ofp53in inbibiting IAV and to screen out the gene(s) that potential regulated by p53, the transcriptomicprofiles of p53+/+and p53-/-mice lung were compared. The result showed that the mRNA of someimmune-related molecules were up-regulated in lungs of p53+/+mice, compared with p53-/-mice, such assome toll like receptor family members and its pathway molecules. And we also found8genes, which maybe poteintial target genes of p53and have roles in anti-influenza virus and the research is under going.Otherwise, we also analyzed the apoptosis and cell cycle pathways, which played important role in theinfection of IAV, and found several genes have expressed obviously differently.Conclusion, we had performed some analysis on the interaction of p53and IAV. The immunity of p53against IAV infection had been demonstrated, including some possible mechanisms, such as cell cycle andapoptosis pathway and some potential p53regulated cellular molecules and immune cells related IAVinfection had been identified. These studies further revealed the innate antiviral role of p53and providedvaluable information for further study of p53anti-IAV mechanism and the development of anti-IAVstrategies.