Genome Wide Screen Identified Host Factors Involved In Avian Influenza Virus Replication And Study On PA Antagonizes Host IFN Signaling

Influenza A virus(IAV)causes acute respiratory infections among humans,poultry and wild birds,makes a great threaten to public health and global economic stability.The high lethality and the potential of cross-species infection of IAV deserve our concern.Notwithstanding,vaccination against seasonal influenza plays an important role in public health system,the rapid spread and recombination of IAV raise a giant challenge to the research and development of vaccine and medicine.Upon infection,the evasion of innate immunity,virus replication and proliferation,inflammatory response must be all associated with the interaction between host factors and virus.Due to the variation of influenza virus during infection and proliferation,the evolution rate of virus against antiviral drugs may higher than that against the drugs that target host factors.Hence,focusing on the virus dependent host factors maybe a new way for seeking the drug targets and therapy of influenza virus.Firstly,we investigated the regulation of host IFN signaling pathway by influenza virus polymerase.We found that the PA subunit of influenza virus could reduce host IFN production.Furthermore,influenza virus PA interacted with host interferon regulatory factor 3(IRF3),suppressed activation and nuclear translocation of IRF3,which leaded to the inhibition of IFN production,and blocked virus-induced host innate immunity response.Next,we performed a genome-wide knockout library based on clustered regular interspersed short palindromic repeats(CRISPR),followed by H7N9 infection,and identified host essential genes that involved in IAV infection.The results were as follows:1.The PA subunit of IAV antagonizes host IFN productionThe PA expression plasmids of different influenza subtypes were generated,including pdm/09 PA(A/Mexico/4486/2009),F9PA(A/swine/Nanchang/F9/2010(H1N1)),H5N1 PA(A/duck/Hubei/Hangmei01/2006(H5N1))and H7N9 PA(A/Shanghai/02/2013(H7N9)).The luciferase assays indicated that these subtypes of PA suppressed IFN-beta promoter activity.Next,we investigated the transcriptional activity of IFN signaling pathways adaptors in the presences of overexpression of PA,result showed that PA could inhibits IRF3 upstream signaling pathways,including RIG-I,MDA5,MAVS and TBK-1,rather than NK-kappa B signaling pathway,which indicated that influenza PA inhibited IFN-beta production through IRF3 dependent-manner.Furthermore,co-immunoprecipitation and indirect immunofluorescence assays demonstrated that PA interacted with IRF3,suppressed IRF3 activation and IFN production.The interaction of PA and IRF3 depend on Asp 108 of PA.2.The screen of host essential genes that involved in H7N9 infectionThe A549 cell were transduced with lentivirus packed genome-wide knockout library to further analysis the interaction between virus and host factors under H7N9 infection.The next generation sequences showed the library with sufficient coverage for subsequent screening.Next,the library cells were subjected with a lethal dose of H7N9 to screen for IAV restriction host factors.Then g DNA extraction,amplification and sequences were performed with survival cells.According to the MAGe CK(Model-based Analysis of Gene Essentiality)analysis score,the top 50 hits knockout polyclonal cell lines were generated for preliminary evaluation.The results showed first 30 hits knockout cell strains slowed down H7N9 proliferation in A549 cell,which indicated the reliability of our generated knockout library.Next,we choose 14 candidate genes knockout cells that siginificant delaed cell death upon H7N9 infection,for generation of monoclonal cell lines,and the 14 knock out cell strains showed suppression of H7N9 infection,remarkably.In the light of IAV life cycle in host cells,the influence of these 14 knock out cell lines on virus binding,internalization,transcription and replication activity were investigated.The results showed,SLC35A1,CMAS,GNE and NANS knockout blocked the binding of IAV,CYTH2 and ABCD2 knockout impaired viral internalization,while SLC35A2,MIIP,CYTH2,RNASE8,HAL-G,ABCD2 and ST18 knockout suppressed the activity of IAV polymerase.This study identified critial host genes that involved in sialic acid synthesis and transport,including SLC35A1,CMAS,GNE,SLC35A2 and NANS.Among these genes,we firstly reported that NANS could involve in IAV propagation,as an essential synthetase of sialic acid pathway.To further confirm these candidate genes functions in IAV replication,we performed retrovirus induced gene complement of CYTH2,TTC24,NANS and H1 FX,and the results showed complement of CYTH2,TTC24 and NANS would recover the H7N9 inhibition by the knockout effects.In summary,the genome-wide knockout library would be a powerful tool for identification of host factors upon virus infection.3.The study of candidate gens CYTH2 and CYTH2 function in IAV endocytosisIn our screen,we found that CYTH2 could significant suppressed H7N9 proliferation and CYTH2 knockout inhibited IAV endocytosis.The retrovirus induced CYTH2 complement of CYTH2 knockout cell recovers the H7N9 inhibition.Meanwhile,CYTH2 inactivated complement cells were still suppressed H7N9 infection with varying degrees.In addition,pretreatment of A549 cells with Secin H3(selective inhibitor of CYTH2)could inhibit H7N9 infection remarkably.These results indicated that full strcture and function of CYTH2 was necessary in IAV infection.With further understanding of CYTH2 knockout on the inhibition of IAV invasion,the IAV particles were labeled with fluorescence dyes and virus invasion was monitored with continuous dynamic observation.The results showed fusion process was blocked in CYTH2 knockout cells,so that suppressed virus uncoating.The electron microscope assay showed,in CYTH2 knockout cells,influenza virions remained in endosome during endocytosis,and could not fusion with endosome,so that suppressed IAV proliferation.Furthermore,CYTH2 knockout also impaired PRV and HSV propagation.These results indicated that CYTH2 was an essentical gene of virus invasion,including RNA virus(IAV)and DNA virus(PRV and HSV),and may be a host potential drug target of various virus infection.