Identification And Biofunction Study Of Host Membrance Proteins Associated With H5N1 Influenza Virus Infection

H5N1 influenza virus is a highly pathogenic avian influenza(HPAI)virus which can infect many kinds of poultry and directly transmit from poultry to humans,bring a great threat to human health.Influenza virus is parasitic,which contains very little genetic information itself.The interaction between the virus and host made the molecular basis of infection,replication,pathogenic and immunization.Because influenza is an enveloped virus,during its infection process,the fusion of the virus and the host plasma membrane,entry and bud of virus particles are critically important steps in the virus life cycle which all rely on the host cellular membrane proteins participation.So,study on the identification of host membrane protein associated with influenza virus infection is of great significance.In this research,we focused on the changes in the levels of membrane proteins of the highly pathogenic avian influenza H5N1 virus infected and the mock infected A549 cells,using comparative proteomics to screen for proteins associated with influenza virus infection and with the responses of human lung epithelial cells to influenza virus infection.Also,we do further studies on the functions of the identified proteins to reveal the pathogenesis of influenza virus.In addition,we also hope to find antiviral drug targets or diagnostic markers from the host and develop new research directions of prevention and treatment of virus disease.The main researches were as follows:1.Identification of the host membrane proteins associated with H5N1 virus infectionIn this research,we focused on the membrane proteins,because of that the membrane proteins play important roles in the influenza virus infection.Membrane proteins extracted from DW-infected and mock infected A549 cells at 6,12,and 24 hpi were separated using 2-DE technology.Comparative analysis of the 2-DE gels revealed 56 differently expression protein spots,among which,20 clearly up-regulated and 36 down-regulated.To identify these proteins in the 2-DE gels,the marked spots were excised and subjected to MALDI-TOF/TOF MS analysis.In all,42 altered protein spots were successfully identified,including 16 up-regulated and 26 down-regulated.Of these proteins,Fifty-seven percent are known or predicted membrane proteins,or proteins known to interact with other membrane proteins.These identified proteins in multi-compartment can engage in different cellular processes,including cytoskeletal components;cell adhesion and interaction;cellular signal transduction;immune response;stress response;transcription/RNA processing and metabolism.2.The influence of the identified proteins on H5N1 influenza viral propagationTo investigate the biological effect of our identified proteins in the influenza virus infection process,we chose six proteins named ANXA2,ClQBP,PHB,PRDX1,HSP90AB1 and ITGA3,to analyze the roles of these proteins in virus propagation.In this research,western blotting and qRT-PCR were used to evaluate the well efficacy siRNAs.The expression of these proteins was substantially inhibited in cells transfected with specific siRNA,then,infected with H5N1 influenza virus.According to the plaque assays of these cell culture supernatants,we found that there are five proteins have effects on the proliferation of influenza virus.Knocking-down endogenous ANXA2 or CkQBP in A549 cells favored the propagation of HPAI H5N1 virus.Moreover,the viral titers in the supernatants were significantly reduced when PHB,PRDX1and HSP90AB1 were silenced relative to the control.However,treatment with siRNA targeting ITGA3 did not result in a significant difference relative to the control.These proteins participated in pathways that are important for antivirus or anti-inflammatory responses.Targeting these proteins or the associated pathways in infected cells will favor the development of novel antiviral therapies.3.Interaction between ANXA2 and influenza NS1 proteinIn this study,membrane proteins extracted from DW-infected and mock infected A549 cells at 24 hpi were analyzed using BN/SDS-PAGE coupled with MALDI-TOF/TOF MS.We found that there are three host proteins can interact with the influenza protein NS1;they are ?-actin,ANXA1 and ANXA2.Also,we conformed the interaction between ANXA2 and NS1 using co-immunoprecipitation and confocal.According to the structure of influenza NS1,we do a further study to truncate the NS1 protein into RNA binding domain and effect domain.Through co-immunoprecipitation analysis,we found that is the effect domain of NS1 protein can interact with ANXA2.This study successfully identified a new host protein interaction with the influenza virus NS1 protein.Also,this study provides a new direction to elucidate the molecular mechanism of influenza virus infection and pathogenic.