Exploratory Study On The Difference Of The Pathogenesis Of H5N1 And H1N1 Subtype Influenza Viruses

IAV is the main pathogen of seasonal influenza epidemic and occasional pandemic among human population.It has widespread host range and variation extension,leaving a heavy threaten to our society.Influenza A virus(IAV)is an enveloped virus in the Orthomyxoviridae family.Its genome consists of eight negative RNA segments,encoding more than ten proteins including Nucleoprotein(NP),Neuraminidase(NA),and Hemagglutinin(HA).Influenza A viruses can be further divided into multiple subtypes according to the antigen specificity of their surface glycoprotein HA and NA.Different subtypes of influenza A virus cause different pathogenic phenotypes when they infect humans.When the virus invades the host,the viral proteins interact with the host proteins to manipulate the host signaling pathways for their own replication and survival.NF-?B signaling pathway is one of the most important antiviral signaling pathways.When pathogenic microorganism infects the human body,the virulence proteins or genetic material of pathogenic microorganism act as ligands to combine with the cell surface receptors,which raises some joint proteins through the structure domain of cell membrane.Then,the joint proteins accumulate and activate the I?B kinase complex(IKK complex).The activated IKK complex will phosphorylate two conservative 32 and 36 serine residues of I?B(Inhibitor of the NF-?B).The phosphorylated I?B proteins will be ubiquitinated and degraded,and then release the nuclear factor kappa B proteins,which are combined with the I?B proteins.The released nuclear factor kappa B is transferred from the cytoplasm to the nucleus and acts on the corresponding target genes to activate or up-regulate the transcription of antiviral genes.The NF-?B signaling pathway is present in almost all animal cells and is a hot issue in the field of signal pathway research.Direct interaction between viral proteins and host proteins is the basis of complex pathology caused by influenza virus invasion.Different subtypes of influenza A virus cause different pathogenic phenotypes after infection in humans because of the different relationship between different subtypes of influenza virus and host signaling pathways.Accordingly,we designed experiments to explore the relationship between influenza virus proteins and NF-?B signaling pathways and the exact sites of interactions to test our hypothesis that different influenza virus proteins interact with human signaling pathways to produce different physiological effects.In our experiment,we made use of two groups of neuraminidase and nucleoprotein of the H5N1 and HIN1 influenza virus to explore the relationship between the NIF-?B signaling pathway and viral proteins by the Luciferase Assay and the potential interaction sites that the viral proteins affect the NF-?B signaling pathway,then we determine the exact sites that the viral proteins affect the NF-?B signaling pathway by the Co-Immunoprecipitation AssayThe specific experimental results are as follows:1.There were differences in the relationship between NP proteins of H5N1 and H1N1 influenza viruses and NF-?B signaling pathwaysThe NP protein of H5N1 influenza virus inhibits the NF-?B signaling pathway at the position of TAK1 and IKKa.The nucleoprotein of H5N1 subtype influenza virus inhibited the transcriptional activity of TNF-a-mediated NF-?B signaling pathway.The results of western blotting showed that the NP protein of H5N1 subtype influenza virus increased the protein expression level of I?B and inhibited the phosphorylation level of I?B proteins.The Immunofluorescence Assays showed that the NP protein of H5N1 subtype influenza virus inhibited the transposition from the cytoplasm to nucleus of the p65 proteins.The results above indicated that the NP protein of H5N1 subtype influenza virus inhibited the NF-?B signaling pathway.The NP protein of H5N1 subtype influenza virus inhibited the transcription activity of NF-?B-luc mediated by TAK complex and IKKa protein,interacted with TAK1 proteins and IKKa and inhibited the phosphorylation of IKKa,indicating that the NP protein of H5N1 subtype influenza virus inhibits the NF-?B signaling pathway at the position of the TAK1 proteins and IKKa proteinsThe NP protein of H 1N I influenza virus has no impact on the NF-?B signaling pathway.The NP protein of HIN 1 subtype influenza virus didn't affect the transcriptional activity of TNF-a-mediated NF-?B signaling pathway,the expression level of I?B protein,the phosphorylation of I?B protein and the nuclear translocation of p65,indicating that the NP protein of H1N1 subtype influenza virus has no effect on the NF-?B signaling pathway2.There were also differences in the relationship between the NA proteins of H5N1 and H1N1 influenza viruses and NF-?B signaling pathwaysThe NA protein of H5N1 influenza virus activates the NF-?B signaling pathway at the position of the TAB2 protein.The deadly H5N1 strain of the influenza virus NA protein increased IL-1?-mediated NF-?B-luc transcriptional activity,suggesting that the deadly H5N1 strain of the influenza virus NA protein can activate the NF-?B signaling pathway.The deadly H5N1 strain of the influenza virus protein NA enhanced TAK complex-mediated NF-?B-luc transcriptional activity,and interacted with the TAB2 protein,suggesting that the deadly H5N1 strain of the influenza virus NA protein activates the NF-?B signaling pathway through the site at the TAB2.The NA protein of the H1N1 subtype influenza virus inhibits the NF-?B signaling pathway at the IKK? site.The NA protein of the HIN1 subtype influenza virus had inhibitory impact on the IL-1?-mediated NF-?B-luc transcriptional activity,which suggests that the H1N1 strain of the influenza virus NA protein can inhibit the NF-?B signaling pathway.The NA protein of the H1N1 subtype influenza virus inhibited TAB2 and IKK?-mediated NF-?B-luc transcriptional activity,and had interaction with IKK? proteins,suggesting that the NA protein of the H1N1 subtype influenza virus inhibits the activity of the NF-?B signaling pathway at the IKK?The comparison between results 1 and 2 verified our hypothesis that the interaction between different proteins of different influenza viruses and human signaling pathways can produce different physiological effects,that is,the interaction between influenza viruses and host proteins has subtype specificity,which can respond to the specific pathogenic mechanism of influenza viruses of different subtypes.Analysis of the results also showed that different subtypes of avian influenza virus could affect the antiviral activity of NF-?B signaling pathway.