Effects Of TRIM38 And TRIM25 On RIG-Ⅰ Pathway-Mediated IFN-Ⅰ Expression

Objective: TRIM(Tripartite Motif)proteins are involved in many important life processes such as cell growth,apoptosis,intracellular signal transduction,autophagy,and tumors.Our previous experiments showed that the levels of most of the TRIM proteins were significantly increased during respiratory syncytial virus(RSV)infection,which shows that TRIM proteins play important roles in this procedure.Existing methods have shown that the RING domain of TRIM proteins have ubiquitin ligase but different functions.When the virus invades in host cells,the body first resists the virus invasion through the innate immune response.Among them,an essential aspect of the innate immune response is the synthesis and secretion of type Ⅰ interferons(IFN-Ⅰ),thereby avoiding the virus from spreading from the infected cell to the adjacent uninfected cell.After the virus invades the cells,different viral genes are recognized and bounded by different receptors in the cells.In this procedure,RLR-like receptors such as RIG-Ⅰ can detect and bind to the viral genomic RNA in the cell,and TRIM25 is a critical protein in the pathway.Futhermore,TRIM25 can induce the production of IFN-Ⅰ and other inflammatory factors by catalyzing RIG-Ⅰ.It has been reported in the literature that TRIM38 inhibits the production of IFN-Ⅰ. RSV is a single-stranded RNA virus that is the leading cause of bronchitis and pneumonia in infants and children.In recent years,RSV is not only prone to sporadic infections,but also may cause large-scale infections,therein leading to the prevalence of respiratory infections worldwide.However,no suitable vaccine for RSV has yet been developed,so the global research on the pathogenesis of RSV has never stopped.Our previous experiments show that human respiratory epithelial cells were infected with RSV and sequenced by whole gene expression.Moreover,the expressions of TRIM25 and TRIM38 were significantly high in these experiments.What role do the two proteins play in the replication of RSV virus? It is known that TRIM25 has K63 ubiquitin ligase activity,while TRIM38 has K48 ubiquitin ligase activity.After RSV infects host cells,it is known that both of them are involved in regulating the production of IFN-Ⅰ,so whether there is an interaction between them in inducing the production of IFN-Ⅰ.Does TRIM38 competitively inhibit the binding of TRIM25 to RIG-Ⅰ? The study of these mechanisms is still unclear.As a result,this study intends to select RSV as a virus model to study the effect of TRIM38 and TRIM25 on the mechanism of IFN-Ⅰ induced by RSV-infected lung epithelial cells,to provide new ideas for the implementation of clinical treatment of RSV.Methods: 1 After A549 cells were infected with RSV,we investigated the effect of TRIM25 and the interferon signaling pathway.1.1 To optimize the multiplicity of infection,we finally selected A549 cells to be infected with RSV-mGFP with a multiplicity of infection of 1(subsequent infection was selected as MOI=1).We chose infection time of 0 h,24 h,48 h,and 72 h.The change in green fluorescence of RSV-mGFP was observed separately.1.2 Infected cells were collected at different time points,and total protein and total RNA were extracted.The expression of TRIM25,RIG-Ⅰ,MAVS,TRAF6,NF-κB,and TRAF3 was detected by Western blot at different time points.The mRNA transcription levels of IFN-α and IFN-β were detected by RT-qPCR.1.3 The constructed plasmid of pCAGGS-HA-TRIM25 was transfected into A549 cells,and the localization of TRIM25 in A549 cells was detected by immunofluorescence.1.4 A549 cells were transfected with the dual luciferase reporter plasmid,and RSV infected the cells for 48 h.Cells were harvested and operated,the activity of the IFN-β promoter was detected by luciferase reporter gene kit.2 TRIM25 induces IFN-Ⅰ production via the RIG-Ⅰ pathway.2.1 The plasmid of pCAGGS-HA-TRIM25 was transfected into A549 cells to overexpress TRIM25 for 24 h.The cells were infected with RSV for 24 h,total RNA was extracted,and mRNA transcription levels of TRIM25,IFN-α,and IFN-β were detected by RT-qPCR.2.2 The Plasmid of pCAGGS-HA-TRIM25 and double luciferase reporter plasmid were co-transfected into A549 cells,and RSV infected for 48 h.The activity of the IFN-β promoter was detected by luciferase reporter gene kit.2.3 In A549 cells,TRIM25 was knocked down by siRNA.When the RSV infected for 48 h,the total RNA was extracted.TRIM25,IFN-α,and IFN-β mRNA transcription levels were detected by RT-qPCR.The total protein was extracted,the knockdown effect of TRIM25 protein level and the expression of RIG-Ⅰ,MAVS,TRAF6,and NF-κB were detected by Western blot.2.4 Pretreatment of A549 cells with an appropriate concentration of IMD0354(IKKβ inhibitor)for 30 min,and add RSV virus for 0 h,12 h,and 24 h.The total protein and total RNA were extracted respectively,and the protein expression of p-P65 and P65 was detected by Western blot.The mRNA transcription levels of IFN-α and IFN-β were detected by RT-qPCR.3 Effects of TRIM38 and TRIM25 on IFN-Ⅰ induced by RIG-Ⅰ pathway 3.1 Overexpression plasmid of TRIM25 transfected into A549 cells.After RSV infection for 24 h,the protein expression of TRIM38 was detected by Western blot.3.2 A549 cells were treated with different doses of human IFN-β for 12 h,and the total RNA was collected.A549 cells were treated with the same dose of IFN-β,total RNA was extracted at different time points,and mRNA levels of TRIM25 and TRIM38 were detected by RT-qPCR.3.3 Vero cells(natural gene defect,no expression of antiviral interferon)overexpressed TRIM25.After RSV infected for 24 h,the protein expression of TRIM38 was detected by Western blot.3.4 In A549 cells,TRIM38 was knocked down by siRNA.When the RSV infected for 48 h,the total protein was extracted.And the expression of TRIM25 and RIG-Ⅰ was detected by Western blot.3.5 The eukaryotic expression plasmid of pCAGGS-HA-TRIM38 was constructed.Different concentrations of plasmid transfected into A549 cells.After RSV infection for 24 h,the total RNA and protein were extracted.TRIM38,IFN-α,and IFN-β were detected by RT-qPCR.The protein expression of TRIM38,TRIM25,and RIG-Ⅰ was detected by Western blot.3.6 A549 cells were co-transfected with TRIM25 plasmid and TRIM38 plasmid with different concentrations for 24 h.Total RNA was extracted,mRNA levels of IFN-α and IFN-β were detected by RT-qPCR.Total protein was extracted,the expression of RIG-Ⅰ,TRIM25,and TRIM38 was detected by Western blot.3.7 The plasmid of pCAGGS-HA-TRIM25 and pCAGGS-HA-TRIM38 co-transfected into A549 cells,and the plasmid of pCAGGS-HA-TRIM25 and Vector transfected respectively.The whole cell lysate of each treated sample was collected to classical magnetic bead immunoprecipitation with RIG-Ⅰ monoclonal antibody.The proteins expression of TRIM25,TRIM38,and RIG-Ⅰ were detected by Western blot.The expression of the above proteins in whole cell lysate was detected.Results:1 The RSV-mGFP infected A549 cells at 0 h,24 h,48 h,and 72 h,the green fluorescence of the cells gradually increased.2 After A549 cells were infected with RSV,the expressions of IFN-α,IFN-β,TRIM25,RIG-Ⅰ,MAVS,TRAF6,and NF-κB in the infected group were significantly higher than those in the uninfected group.The luciferase reporter gene showed that the IFN-β promoter activity was also significantly higher than that of the Mock group(P=0.0203).The expression level of TRAF3 was not significantly different from that of the uninfected group.3 A549 cells overexpressing TRIM25 were infected with RSV for 24 h,the expression levels of TRIM38,IFN-α,and IFN-β in the overexpression group were significantly higher than those in the Mock group.The results of the dual luciferase reporter gene also showed that the activity of the IFN-β promoter in the overexpression group was significantly higher than that in the Mock group(P=0.0145).4 TRIM25 was knocked down in A549 cells.After RSV infection,the mRNA levels of IFN-α,IFN-β,RIG-Ⅰ,MAVS,TRAF6,and NF-κB in the knockdown group were lower than those in the Mock group.5 IKKβ inhibitor pretreated A549 cells.After RSV infection,as the expression of p-P65 was inhibited,the expression levels of IFN-α and IFN-β were significantly lower than those of the Mock group.6 Different doses of human IFN-β treated A549 cells,and the same dose of IFN-β treated A549 cells at different times,the expression levels of TRIM25 and TRIM38 were significantly increased in a dose-dependent manner.7 Vero cells overexpressing TRIM25 were infected by RSV,and there was no significant difference in protein expression of TRIM38 between overexpressing TRIM25 group and Mock group.8 TRIM38 was knocked down in A549 cells.After RSV infected the cells,RIG-Ⅰ protein expression level was significantly higher than the Mock group.9 A549 cells were transfected with different concentrations of TRIM38 plasmid.After RSV infected the cells,we examined that TRIM38 increased with the increase of plasmid concentrations.Compared with the Mock group,the expression of IFN-α and IFN-β in the overexpression group were decreased(P<0.05).The protein expression of RIG-Ⅰ also showed a decreasing trend.10 After co-transfection the plasmid of pCAGGS-HA-TRIM25 and the increasing concentrations of the plasmid of pCAGGS-HA-TRIM38 to A549 cells,the expression of IFN-α and IFN-β in the co-transfection group showed a decreasing trend(P<0.05).The protein expression of TRIM38 and RIG-Ⅰshowed a decreasing trend.11 RIG-Ⅰ can interact with TRIM25 and TRIM38 simultaneously.In the presence of TRIM38,RIG-Ⅰ combined with TRIM25 was reduced.Conclusion:1 RSV-infected A549 cells induced TRIM25 expression,and TRIM25 may mainly induce IFN-Ⅰ production by RIG-Ⅰ activation of NF-κB pathway.2 TRIM25 induces the expression of IFN-Ⅰ,which feedback induces the expression of TRIM25 and TRIM38.3 When TRIM38 coexists with TRIM25,TRIM38 competes with TRIM25 for binding to RIG-Ⅰ,thereby inhibiting the expression of IFN-Ⅰ.