Proteomics Of CEF Infected With H5N1 NS1 EIF4GI Binding Domain Truction Virus And Mechanism Of HNRNPK Promote Influenza Virus Proliferation

Avian influenza virus which has a wide range of hosts and not only infect various poultry,cause significant economic losses to the poultry industry,but also infect humans occasionally,severely impair human life and health because of the high mortality caused by infection.The survival of influenza virus strictly depends on the host cell,and the interacts with various of host proteins is necessary for the infection,replication,pathogenicity and evasion of host immunity of virus.The non-structural 1 protein is a key virulence factor that regulates the replication of influenza virus.The NS1 protein is an important virulence factor that regulates virus replication through a variety of strategies to evade the host immune response.The amino acid 81-113 of the NS1 protein recruits the host protein eukaryotic translation initiation factor 4GI(eIF4GI)to the 5’ untranslated region of the viral m RNA,and preferentially translate the viral m RNA.Our previous study demonstrated that the recombinant H5N1 avian influenza virus lacking the NS1 e IF4 GI binding domain(rNS1-SD30)showed significantly low virulent and pathogenic in both in vitro and in vivo experiments,compared to the wild virus(rNS1-wt),and the ability to inhibit IFN-β on MDCK cells is also impaired.The changes in the host proteins caused by recombinant rNS1-SD30 infection and the pathogenic mechanisms in which those host factors are involved in the reduction of virulence and pathogenicity are worth for further study.Heterogeneous nuclear ribonucleoprotein(HNRNPs)family proteins have many functions,mainly involved in the regulation of m RNA splicing,transcription,stabilization,translation and transport,and some proteins involved in host immune response regulation.In recent years,more and more studies have been done on the regulation of viral replication through regulating the splicing of viral m RNA and interacting with viral proteins by HNRNPs,while HNRNPK participates in the replication of multiple viruses.NS1-BP together with HNRNPK directly bind to the M1 m RNA of influenza A virus to promote the splicing of M1 m RN A to produce M2 m RNA,resulting in increased production of encoded M2 protein,thereby facilitating the replication of influenza virus.However,it is unclear whether the interaction of HNRNPK with influenza virus proteins and whether it participates in the regulation of host innate immune.In this study,comparative proteomic methods were used to study the differential expression of host proteins of chicken embryo fibroblast(C EF)cells infected with rNS1-wt and rNS1-SD30 viruses,and studied the effect of AN XA7 on the replication of virus and the regulation of poultry innate immune response,and also studied the interaction of human HNRNPK with influenza virus and the role of HN RNPK in IFN regulation.The main research contents and results of this study are as follows:1.Proteomics study of CEF cells infection by rNS1-wt and rNS1-SD30 virusesAfter C EF cells infection with rNS1-wt and rNS1-SD30 for12,24,and 36 h,81 differentially expressed host proteins were identified by using Two-dimensional electrophoresis and MALDI-TOF-MS technology.Functional classification analysis of the identified proteins revealed that these differential proteins were mainly involved in the cytoskeleton,apoptosis and stress responses,transcriptional regulation,m RNA processing and splicing,transport and metabolism processes,and cellular signal transduction.And then cloned and expressed part of the genes and studied its effect on virus replication,we found that ANXA7 inhibited the replication of rNS1-SD30,but had no effect on rNS1-wt in DF1 cells.2.ANXA7 regulates the expression of MDA5 to promote chicken RLR signaling pathwayWe constructed the chicken RLR signaling pathway research gene and found that ANXA7,ALDH7A1,and DCTN2 showed strongly enhanced IFN-β promoter activity induced by MDA5.Further study showed that ANXA7 promoted IFN-β promoter activity stimulated by rNS1-SD30 virus,without affecting the IFN-β promoter activity induced by rNS1-SD30 virus.ANXA7 promotes IFN-β production induced by poly I:C and MDA5 by targeting to MDA5 and regulating its protein expression.Moreover,ANXA7 interacts with MDA5,and its C-terminal calcium ion and phospholipid binding region is important for promoting IFN-β production.3.Human HNRNPK promotes influenza virus replication by interacting with NS1 proteinHNRNPK was down-regulated after A549 cells were infected by influenza virus.The overexpression of HNRNPK significantly promoted the proliferation of multiple subtype influenza viruses,and the silence of HNRNPK inhibited virus proliferation.Furthermore,we found that HRNPK interacts with NS1 in an RNA-independent manner.The N-terminal RBD of NS1 and the RNA-binding domain KH2 and protein-protein interaction region KI of HNRNPK are crucial for the interaction between NS1 and HNRNPK.The KH2 domain and nuclear shuttle domain KNS of HNRNPK are key regions for the promotion of HNRNPK on the proliferation of influenza virus.4.HNRNPK inhibits IFN-β production and involved in the innate immunity evasion of NS1Our study found that HNRNPK inhibits IFN-β production and phosphorylation of IRF3 induced by influenza virus and Sendai virus(Sev).Further studies revealed that HNRNPK interacts with TRAF3 of RIG-I signaling pathway and the two RNA-binding domains KH1 and K H2 and protein-protein interaction region KI of HNRNPK and the RING finger domain of TRAF3 play a key role in the interaction between TRAF3 and HNRNPK.In addition,we found that HNRPK enhances the inhibited of NS1 protein on the phosphorylation of IRF3 induced by influenza virus,and the presence of HN RNP facilitates the formation of the NS1-TRAF3 complex.