Characterise Of The Interaction Betweeniav-NEP And Cellular Protein GPS2

Influenza A virus is an important zoonotic pathogen,can infect many species,including person,swine and chicken.Attributed to its high variation and infectiousness,every year it posing a serious threat to the world's human health.In order to well prevent and control this disease,Scientists have been trying to study the pathogenic mechanism of the pathogen.When influenza A virus infected host cells,it replicates in the cell,and then mang protein-interations happen during its life cycle.NEP is coded by NS segment of influenza A virus through RNA splice,known as mediating the export of vRNPs from the host cell nucleus.Protein sequences blastn shows that NEP is very conservative among different subtypes of influenza A viruse.So far,besides mediates the nuclear export of vRNP,no other function has been described.In the previous study,we set NEP as bait protein,and have selected an interaction factor GPS2 from human cDNA library through yeast-two-hybrid.In this study,we aim to validate the interaction between NEP and GPS2,and then describe some biology roles of the interaction.The principal research is decribed as fellow:(1)Validating the interaction between NEP and GPS2Firstly,we validate the interaction through mammalian-two-hybrid.NEP and GPS2 are cloned into pBind-vector and pACT-vector,respectively.Next pACT-GPS2 and pBind-NEP are co-transfected into COS-1 cells.It suggests that the interaction between NEP and GPS2 is really existed.Furthermore,we clone the eukaryotic expression vector pcDN A3.1-c-My c-NEP,pCMV-Tag2B-GPS2,and prokaryotic expression vector pGEX-6p-1-NEP,pET41a-GPS2.Next we do the co-immunoprecipitation and GST pull-down experiments.They all demonstrate that NEP can interact with GPS2 in vivo and in vitro,resprctively.Also,we have studied the celluar location of two proteins in hela cells.when transfect GPS2 alone,GPS2 is mainly located in nuclear.Contrast to that,when GPS2 cotransfected with NEP,we find that GPS2 is transported to the cytoplasm,and colocalized with NEP in cytoplasm.Consequently,we can confirm that NEP interacts with GPS2.(2)The conservative study of the interaction between NEP and GPS2NEP is conservative protein in the influenza A virus.But among viruses of different subtypes still exist some acid sequence differences.We cloned the coding nucleotides of NEP from A/WSN/1933(H1N1),A/Mexico/001/2009(H1N1),A/duck/Hubei/Hangmei/01/2006 into pBind-vector,respectively.The results of mammalian two-hybrid prove that,in spite of the acid sequence differences among these NEPs,they all could interact with GPS2.(3)Mapping the interaction domain between NEP and GPS2According to previously research work,as well as the NEP protein structural characteristics,we have constructed a lot of deleted-mutations of NEP,including NEP(2-llaa),NEP(12-21aa),NEP(22-31aa),NEP(32-41aa),NEP(42-53aa),NEP(2-53aa),NEP(54-121aa),NEP(12-121aa),NEP(20-121aa).They all cloned into pBind-vector.And then we test the interactions with GPS2 through mammalian-two-hyhrid,respectively.It suggests that the interaction domain in NEP is the peptide 32-41aa.Also we have study that the 32-41aa peptide is a nuclear export signal.(4)GPS2 impacts the replication of influenza A virusFirstly,we used the mini-replicon system to test the polymerase activity of influenza A virus,and study whether overexpression GPS2 inpacts the polymerase activity in 293T cell.It suggests that when overexpressed GPS2,the polymerase activity of influenza A virus is activated.Next,when co-expressed the NEP,it can alleviate the activation to the polymerase activity of influenza A virus.(5)GPS2 reduce the ativation of AP-1 dependent genes expression by replication of influenza A virusIn Hela cells,the expression of GPS2 can inhibit the expression of AP-1 dependent genes,which is activated by TNF-a.Also,when GPS2 is mainly located in cytoplasm,its inhibition efficiency is much more apparente.In MDCK cells,GPS2 can inhibit the expression of AP-1 dependent genes activated by influenza A virus infection.