The Study On The Molecular Mechanism Of Tembusu Virus NS5 Protein Antagonizing Type ? Interferon Signaling

Since the outbreak in 2010,Tembusu virus(TMUV)infection has spread to most duckrearing regions rapidly and seriously impacts the development of aquaculture in China.The causative pathogen of this disease is TMUV,which belongs to the family Flaviviridae and genus Flavivirus and it causes paralysis in ducklings and heavy egg drop in laying ducks.As the first line of protecting the host from pathogenic organisms,the innate immune response plays an important role in inducing an antiviral state in cells to curb viral replication and dissemination.Type I interferon(IFN-I)system,as the crucial part of the innate immune response is widely studied those years and it has been determined that the nonstructural(NS)proteins of TMUV could interact with different adaptor proteins,resulting in the inhibition of IFN-I induction.Therefore,in order to further reveal the relationship between TMUV and the innate immune response,a series of researches were performed to analyze the inhibitory effect of TMUV on JAK-STAT signaling,and we identified that NS5 protein was responsible for blocking ISGF3 nuclear translocation.The present study now will provide an explanation of the innate immune suppression induced by TMUV and the results were shown as below: 1.TMUV negatively regulates IFN-I signalingIFN-I needs to be recognized by the Interferon ?/? receptor(IFNAR)and then play the antiviral effects after being transduced by the JAK-STAT signaling pathway.In this study,to analyze whether IFN-I can effectively inhibit virus infection,the cells were firstly infected with TMUV(0.2,1 MOI),then stimulated using a certain dose of IFN-?(50,500 U/m L).However,via IFA and real-time PCR assay,the viral expression levels were not affected.The data above indicated that TMUV may have the inhibitory abilities to the IFN-I immune response.Interferon-stimulated gene(ISG)is the performer of antiviral effects of IFN-I,and its expression is mainly regulated via IFN-stimulated response element(ISRE).Next,we analyzed the related ISGs(ISG15,ISG56,Mx1 and OAS1)expression levels and ISRE promoter activation in TMUV-infected cells(0.2,1 MOI),and after stimulation with IFN-?(500 U/m L),these tested indicators were decreased significantly compared with mock.Subsequently,vesicular stomatitis virus(VSV)was used to confirm the results above and the proliferation of VSV was significantly suppressed in the IFN-?(500 U/m L)treatment group,while there was little effect in TMUV-infected cells(1 MOI).Based on these results,we determined that TMUV could negatively regulate IFN-I signal transduction.2.Molecular mechanism of TMUV-NS5 blocking IFN-I signalingSome researches about flavivirus have demonstrated that the NS proteins play an important role in viral replication,translation and the inhibition of host innate immunity.Therefore,in this study,NS1,NS2 A,NS2B,NS3,NS4 A,NS4B and NS5 eukaryotic expression plasmids were co-transfected with luciferase reporter plasmid(p ISRE-TA-luc)and internal reference plasmid(p RL-TK)into HEK293 cells.After IFN-?(1000 U/m L)treatment,luciferase assay and realtime PCR test were performed respectively,and the results showed that the ISRE activity as well as the subsequent ISGs expression were significantly suppressed by TMUV-NS5.It is known that IFN-I binds to IFNAR which located on the surface of cell membrane and phosphorylates the key transcription factors in the JAK-STAT pathway.Then,the interferon stimulated gene factor 3(ISGF3)which formed by phosphorylated signal transducer and activator of transcription(p STAT1,p STAT2)and interferon regulatory factor 9(IRF9),translocates to the nucleus where it bands to ISRE and promotes the transcription of ISGs.In the present study,the protein levels of key factors in the JAK-STAT signaling pathway were detected in NS5-expressied HEK293 cells and after IFN-?(1000 U/m L)stimulation,expression levels of Janus kinase(JAK1),IFNAR1,STAT1,STAT2 and IRF9 were not affected by TMUVNS5 and it also did not interfere with the phosphorylation of JAK1,STAT1 and STAT2.Moreover,co-Immunoprecipitation(co-IP)tests showed that NS5 protein did not interact with STAT1/STAT2 and the formation of ISGF3 complex was not blocked.However,the nuclear localization of STAT1/STAT2 was decreased in NS5 affected cells.These data suggested that the inhibitory effect of TMUV-NS5 on STATs nuclear translocation is the main reason for IFNI signaling suppression.3.The inhibition mechanism of ISGF3 nuclear translocation by TMUV-NS5It has been known that after phosphorylation,p STAT1 requires the assistance of Importin ?5(also known as KPNA1)to transport to the nucleus.In this study,empty vector or HA-NS5 eukaryotic expression plasmid was co-transfected with Myc-KPNA1 into HEK293 and Hela cells respectively.After stimulated by IFN-?(1000 U/m L),co-IP tests revealed that TMUVNS5 hindered the interaction between KPNA1 and p STAT1,which led to the inhibition of ISGF3 nuclear translocation.Subsequently,we also found that TMUV-NS5 did not reduce the protein levels of karyopherins,but it could be interacted with KPNA1,which was the key factor for the nuclear translocation of p STAT1.Furthermore,in HEK293 cells which overexpressed with KPNA1,the replication levels of TMUV were significantly inhibited.According to the above results,we confirmed that TMUV-NS5 could block the transport of ISGF3 into nucleus by interacting with KPNA1,thus antagonizing IFN-I signal transduction.4.Analysis of the interaction area between TMUV-NS5 and KPNA1Nuclear localization signal(NLS)is an important region that recognized by KPNA in the cargo proteins(generally larger than 40 k Da).In the present study,two potential NLS regions were predicted as possible domains that responsible for the interaction between TMUV-NS5 and KPNA1.NLS-P1(28?46 aa),which located at the N terminal of NS5 protein sequence,was predicted by different methods.Moreover,this sequence was identified existing on the surface of the three-dimensional structure of TMUV-NS5,which increases the possibility of KPNA1 recognition and NLS-P1 2×EGFP completely appeared in the nucleus,indicating that it has a strong activity.Another one,NLS-P2(369?405 aa),which was widely studied in flavivirus(such as DENV,ZIKV)contains key sites recognized by KPNA,but there has some difference in amino acid sequences among various flaviviruses.Furthermore,for getting more intuitive results,we constructed the mutant expression plasmids of TMUV-NS5 including HANS5(Contain NLS-P1),HA-NS5(Contain NLS-P2)and then co-transfected with Myc-KPNA1 into HEK293 cells.Co-IP tests showed that the mutants of NS5 protein deleted NLS-P1 could weaken its interaction ability with KPNA and via further truncation in NLS-P1 region,we confirmed that the sequence in N terminal of TMUV-NS5(37?46 aa)was the key region that mediates its interaction with KPNA1.