Studies On The Mechanism Of DEAD-box RNA Helicase 18 Inhibits IFN-? Production

The transcription factor IRF3(Interferon regulation factor 3)plays a very imp ortant role in the production of type I interferon(IFN).TLRs(Toll-like receptors)and RLRs(RIG-I-like receptors,RLRs)mediate the production of type I interferon by activating IRF3 and the binding of IRF3 to the transcription factor binding sit es on the promoter of type I interferon,then induce the production of type I inter feron.The pathway of interferon production is carefully regulated,and there are m any molecules in the host to regulate the production of type I interferon.Previousl y,there have been a lot of reports about how host factors or pathogen-coding prot eins regulate IRF3 activation,but most of the studies focused on the regulation of IRF3 activation in cytoplasm.There are few reports about how the molecules in nucleus regulate IRF3,especially the negative regulation of IRF3.In order to expl ore the mechanism of molecular regulation of IRF3 in the nucleus,we screened th e nuclear proteins interacting with IRF3,confirmed that DDX18 interacted with IR F3 and negatively regulated the activation of IFN-beta promoter mediated by IRF3.On this basis,we studied how DDX18 negatively regulated IRF3-induced IFN-bet a production.The main research contents and results are as follows:1.IP-MS of IRF3 in nucleusIn order to identify the proteins interacting with IRF3 in the nucleus,the euk aryotic expression plasmid and the corresponding empty vector plasmid of IRF3 w ere transfected into HEK-293 T cells respectively.The nucleoprotein was extracted and immunoprecipitated with antibodies against IRF3 protein and analyzed by mass spectrometry.Several cell proteins including DDX18(DEAD-box helicase 18),N COA5(nuclear receptor coactivator 5)and AHNAK(AHNAK nucleoprotein)were screened for potential interaction with IRF3.Considering that DEAD-box RNA he licase family proteins are extensively involved in RNA metabolism and natural im mune regulation,and there are no reports on the regulation of interferon productio n by DDX18,this topic chooses DDX18 for further research.2.Verification of interaction between DDX18 and IRF3To verify the interaction between IRF3 and DDX18,DDX18 and IRF3 eukary otic expression plasmids were co-transfected into HEK-293 T cells.The results of i mmunoprecipitation confirmed the interaction between DDX18 and IRF3.In order to analyze the specific regions of interaction between DDX18 and IRF3,truncated mutants of N-terminal,C-terminal and helicase core regions of DDX18 were cons tructed.The eukaryotic expression plasmids of IRF3 and DDX18 truncated bodies were co-transfected into HEK-293 T cells,respectively.Immunocoprecipitation assay showed that IRF3 interacted with the core region of DDX18.3.DDX18 negatively regulates IFN-beta productionDDX18 interacts with IRF3,and IRF3 is the key transcription factor regulatin g IFN-beta production.Therefore,it is speculated that DDX18 may play a role in regulating IFN-beta.Different doses of DDX18 eukaryotic expression plasmids we re co-transfected with p RL-TK and IFN-beta-Luc into HEK-293 T cells.Then the e ukaryotic expression plasmids of IRF3 were stimulated or re-transfected with Send ai virus(Se V).The results showed that DDX18 protein inhibited the activation of IFN-beta promoter induced by Se V and overexpression of IRF3 in a dose-depende nt manner.At the same time,si RNA interfered with the expression of endogenous DDX18.It was found that interfering with the expression of DDX18 could signif icantly up-regulate the activation of Se V and IFN-beta promoter induced by overex pression of IRF3.In order to further confirm that endogenous DDX18 has the fun ction of negative regulation of IFN-beta,DDX18 knockout cell lines were construc ted by CRISPR-Cas9 technology.By comparing the activity of IFN-beta promoter in wild-type and knockout cell lines stimulated by SEV and over-expression of IR F3,it was confirmed that knockout DDX18 can significantly up-regulate the activa tion of IFN-beta promoter.4.Negative regulation of IFN-beta by DDX18 does not depend on the activity of its helicase and ATPaseBy comparing the conservative motifs of DEAD-box family members,ATPase mutants DDX18-K229 A and helicase mutants DDX18-S364 L were constructed.Th e eukaryotic expression plasmids of wild DDX18 and DDX18-K229 A and DDX18-S364 L were transfected into HEK-293 T cells respectively.The results showed that the two mutants of DDX18 still inhibited the activation of IFN-beta promoter indu ced by Se V and overexpression of IRF3,and the inhibition effect was similar to t hat of wild DX18.Further,the wild DDX18 or mutant DDX18-K229 A and DDX18-S364 L were knocked out in DDX18 cell lines.It was found that both wild DX18 and mutant DDDX18 could restore the level of IFN-beta promoter activation.Th ese results indicated that DDX18 negatively regulated IFN-beta activity independent of its helicase activity and ATPase activity.5.DDX18 blocks the binding of IRF3 to IFN-beta promoterIn order to further study the specific mechanism of DDX18 inhibiting IFN-bet a production,DDX18 was co-expressed with RIG-I,MDA5,IPS-1,TBK1,IKK ep silon and IRF3,which were key molecules in RLRs signaling pathway.It was fou nd that DDX18 could significantly inhibit the activation of IFN-beta promoter indu ced by the above molecules,and DX18 could also inhibit the activation of IFN-be ta promoter mediated by IRF3-5D dominant mutant,confirming that DX18 played an inhibitory role as IRF3-5D promoter.The target is indeed located in the nucleu s.Considering the interaction between DDX18 and IRF3 in the nucleus,it is spec ulated that DDX18 inhibits the binding of IRF3 to IFN-beta promoter by interactin g with IRF3.To confirm this hypothesis,the effect of DDX18 overexpression on t he binding of IRF3 to IFN-beta promoter was analyzed by chromatin immunopreci pitation method.The results showed that both DDX18 and its helicase mutants co uld inhibit the binding of IRF3 to IFN-beta promoter,but DDX18 could not direct ly bind to IFN-beta promoter.