Screening,Identification And Functional Analysis Of The Host Proteins That Interact With The Highly Pathogenic H5N1 Avian Influenza Virus PA Protein

Since the first report of human case in Asia in 1997,H5N1 influenza viruses have rapidly spread to more than 63 countries in Asia,Europe,and Africa.The AIV causes acute respiratory diseases.It not only infects poultry,causing enormous economic losses,but also infects and kills mammals,including humans,posing a great threat to public health.The genome of the influenza virus contains 8 RNA segments encoding more than 17 viral proteins.The PA protein is the third subunit of viral polymerase complex and plays a key role in the life cycle and pathogenicity of the influenza virus.Firstly,the N-terminal of PA possesses the endonuclease activity that impacts the viral transcription and replication.Secondly,the key amino acid of PA protein is an important virulence factor of influenza virus,which can affect the pathogenicity of the virus and host adaptation.Thirdly,the PA gene also encodes three novel proteins,including PA-N155,PA-N182 and PA-X,which play an important role in influenza virus infection.Because of the limited genetic coding capacity,viruses heavily rely on the host cell machineries for their life cycle.The extensive protein-protein interactions between influenza virus and host are extremely important for the infection,replication,and spread of influenza viruses and the resistance of host to IAV infection.Considering the key role of PA protein and virus-host protein interaction in viral lifecycle,exploring host factors that interact with PA protein is a crucial manner to accelerate our understanding on the mechanism of the influenza virus infection.In this study,we selected a H5N1 avian influenza virus(CK10)that showed high pathogenicity in mice to explore host proteins interacting with PA.We screened 278 host cellular proteins that may interact with the PA protein.Among these host proteins,we identified NCL and eEF1A1 are able to interact with PA protein.Moreover,these two proteins were also identified in other subtype viruses as potential influenza virus host interacting proteins.In order to further screen the key host proteins that interact with PA,we subsequently selected a strain of H5N1 avian influenza virus(GS10)that showed low pathogenicity in mice to explore different host proteins interacting with PA from CK10 and GS10.The different pathogenicity between CK10 and GS10 in mice was mainly determined by PA.Compared with the low pathogenic GS10,there were 160 host proteins interacting with the high pathogenic CK10 specifically,including eEF1A1.NCL and eEF1A1 have multiple biological functions.NCL possesses various molecular functions,such as intrinsic self-cleaving,DNA helicase,RNA helicase and DNA-dependent ATPase activity.Meanwhile,NCL participates in various kinds of biological processes,including ribosome biogenesis,cytokinesis,nucleogenesis,cell proliferation and growth,cytoplasmic-nucleolar transport of ribosomal components,transcriptional repression,replication and signal transduction.The eEF1 A1 is not only a translation factor but also a pleiotropic protein,which modulates cytoskeleton,exhibits chaperone-like activity and also controls cell proliferation and cell death.Considering the multiple functions of NCL and eEF1A1,we then analyzed the effects of NCL and eEF1A1 on influenza virus and found that NCL and eEF1A1 played an antiviral role in CK10 virus infection.1.Screening and identification of host proteins interacting with the CK10 virus PA proteinAccumulating data have identified the important roles of PA protein in replication and pathogenicity of AIV.Identification of host factors that interact with the PA protein may accelerate our understanding of IAV pathogenesis.In this study,using immunoprecipitation(IP)assay combined with liquid chromatography-tandem mass spectrometry in CK10 infected A549 cells;we identified 278 human cellular proteins that might interact with PA of H5N1 IAV.Gene Ontology(GO)annotation revealed that the identified proteins are highly associated with viral translation and replication.Further KEGG pathway analysis of the interactome profile highlighted cellular pathways associated with translation,infectious disease and signal transduction.In addition,Diseases and Functions analysis suggested that these cellular proteins are highly related with Organismal Injury and Abnormalities and Cell Death and Survival.Moreover,two cellular proteins,nucleolin(NCL)and eukaryotic translation elongation factor 1-alpha 1(eEF1A1)identified both in this study and others were further validated to interact with PA using co-immunoprecipitation and co-localization assays.Therefore,this study presented the interactome data of H5N1 IAV PA protein in human cells which may provide novel cellular target proteins for elucidating the potential molecular functions of PA in regulating the lifecycle of IAV in human cells.However,further studies are still needed to explore the potential roles of these two proteins in the life cycle of IAV.2.Screening the different interactions between PA protein from two H5N1 avian influenza viruses with markedly different pathogenicity in mice and host proteinsTo further explore the host factors that contribute to the pathogenicity of the H5N1 influenza virus,we selected two H5N1 avian influenza viruses with different pathogenicity in mice mainly determined by PA(CK10 and GS10).A549 cells were infected with these two viruses and IP experiments were performed by using PA antibodies subsequently to screen host proteins that interact with PA.The host proteins were further identified by LC-MS/MS.We found that there were 160 host proteins including the previously identified eEFIA1 protein interacting with the high pathogenic CK10 specifically when compared with the low pathogenic GS10.Then we analyzed these 160 proteins through bioinformatics analysis.GO annotation analysis showed that these proteins mainly involved in the biological processes of translation,gene expression,viral transcription and viral infection.Through the KEGG pathway analysis,we found that these proteins mainly participate in the cell pathways of translation,infectious diseases and signal transduction.Therefore,this study successfully screened out the differential interactome data of PA protein from different pathogenicity H5N1 influenza viruses.Our study may not only play pivotal roles in elucidating the pathogenesis of H5N1 influenza virus,but also might provide new direction for the development of anti-influenza drugs.3.Effect of PA interacting protein eEF1A1 on influenza A virus infectionInfluenza virus heavily relies on the host cell machineries for their life cycle.EEF1A1 is not only a translation factor but also a pleiotropic protein,exerting cytoskeleton modulation,chaperone-like activity,and regulation of cell proliferation and cell death.Since we have indentified eEFlAl as a new PA interacting partner,we then explored the effect of eEF1A1 on the infection of AIV.We found that inhibition of the expression of eEF1Al significantly increased the RNP activity,mRNA,cRNA and vRNA synthesis,replication efficiency of CK10 and significantly enhanced viral induced cytokine response.Moreover,we also found that the nuclear accumulation of viral polymerase component was obviously increased in sieEF1A1 Cells.On the contrary,overexpression of eEFlA1 resulted in reduced replication efficiency of CK10.Moreover,eEF1A1 also showed an inhibitory effect on H5N1 and H7N9 replication,suggesting a broad inhibitory effect of eEF1A1 on various influenza viruses.Furthermore,eEFlA1 had no effect on IAV induced apoptosis,while promoted virus induced necrosis.RNA-seq analysis showed that depressed the expression of eEF1Al resulted in up-regulating the genes associated with chemokine pathway,influenza A pathway,transcription and negative regulation of cell death.And the results of RNA-seq analysis were consistent with the effects of eEFlA1 on inhibiting of viral replication,reduction of chemokines and promoting of cell necrosis during CK10 infection.Therefore,we suppose that eEFlA1 may act as an antiviral factor in AIV infection and may play an important role in necrosis induced by IAV.4.Effect of PA interacting protein NCL on influenza virus infectionOn one hand,IAV is a nuclear replicating virus which has direct access to and hijacks host nuclear for the purpose of the optimum infection.Transportation of the influenza viral RNP complexes into the nucleus is obligatory for virus hijacking the host cell machinery and the subsequent viral replication.On the other hand,NCL predominantly locates at nucleolus and disadvantageously distribute over nucleoplasm,cytoplasm and cell surface.Therefore,considering the significant molecular functions of NCL in multiple biological processes,including ribosome biogenesis,DNA repair and replication,cytokinesis,virus infection and RNA transcription,it is highly probable that NCL may associate with viral RNP activity and virus propagation.Based on this hypothesis,we surmised that NCL-PA interaction may play an important role in IAV infection and proliferation.The above study has demonstrated that nucleolin(NCL)is a novel PA-interacting host protein.Therefore,we further explored the potential role of NCL during the highly pathogenic H5N1 avian influenza virus infection.We found that depletion of NCL in mammalian cells by siRNA targeting endogenous NCL resulted in significantly increased viral polymerase activity,elevated viral mRNA,cRNA and vRNA synthesis,accelerated viral replication and enhanced cell apoptosis and necrosis during H5N1 virus infection.Moreover,siRNA of NCL also significantly exacerbated the inflammatory response,including IL-6,TNF-?,TNF-? CCL-4,CCL-8,IFN-a,IFN-? and IFN-,y.On the contrary,overexpression of NCL significantly decreased the viral replication.Collectively,this work identified that NCL as a potential novel antiviral factor during H5N1 infection,further studies exploring the relevant antiviral mechanism of NCL may accelerate the development of new anti-influenza drugs.