TRIM22 Regulated RSV Replication Through Autophagy

Respiratory syncytial virus?RSV?is one of the most important pathogens causing respiratory viral infection in infants and young children,especially acute lower respiratory infection?ALRI?,and it is an important cause of hospitalization in infants and young children.A meta-analysis estimated that22 % of children with ALRI under 5 years of age are attributable to RSV.The study of the mechanism of RSV infection is helpful for the exploration of RSV prevention and new treatments.TRIM?Tripartite motif?protein family is a large family with more than80 members,most of which have characteristic RBCC domains and possess E3 ubiquitin ligase activity,a feature that is believed to contribute significantly to their functions.TRIM proteins are involved in many life processes.and their performance in virus infection and autophagy have attracted much attention.Experimental studies have shown that TRIM proteins participate in the process of virus and non-virus-induced autophagy,and TRIMs play a key role in the process of autophagy.Autophagy is a complex cellular metabolic process which is highly conserved.It phagocytoses damaged or redundant proteins,organelles,or pathogen derivatives through a double-membrane structure to form autophagosomes and target lysosomal to degrade.Autophagy is associated with the occurrence of various diseases such as tumors,aging,neurodegenerative diseases and infections.Many different viral infections can induce autophagy,and the effects of autophagy on virus proliferation are quite different.TRIM proteins plays an important role in the process of virus and non-virus-induced autophagy,suggesting that TRIM plays a key role in the process of autophagy.The purpose of this subject was to study the role and possible mechanism of TRIM proteins in RSV infection of respiratory epithelial cells,and to revealthe role of TRIM proteins in RSV infection and host cells,and to provide a theoretical basis for the prevention and treatment of RSV infectious diseases.Part one Expression of TRIM Proteins in RSV Infected A549 CellsObjective: To investigate the effect of RSV on the expression of TRIM proteins in A549 cells.Methods:1 RSV infected A549 cells with MOI=5 for 24 h,and transcriptome sequencing was used to detect the expression of TRIM proteins.2 RSV infected A549 cells for 24 h.According to the results of the previous step,the TRIM proteins with different expression between the infected group and the control group were selected.RT-qPCR experiments were performed to verify the differential expression of TRIM proteins mRNA.3 Total proteins were extracted at 0 h,6 h,12 h,24 h,36 h,48 h in infected A549 cells.The expression of TRIM22,14,21,25 were detected by Western blot.4 RSV infected A549 cells for 48 h,and immunofluorescence staining was performed to observe the positive expression and localization of TRIM22 in cells.Results:1.According to transcriptome sequencing analysis of infected A549 cell,a total of 333 differential genes were screened.Compared to the control group,there were 229 genes upregulated and 104 genes down-regulated in the infection group.The screening criteria for the fold fold> 2,q <0.05.There were five differential genes in the TRIM proteins family,which were TRIM22,14,21,25 and TRIM38,and all were up-regulated in the infected group.Among them,TRIM22 has the largest difference of 3.03 times.2.The different expression of TRIM22,14,21,25 and TRIM38 were verified by RT-qPCR.The results confirmed that TRIM22,14,21,25 and TRIM38 were higher after infecting with RSV for 24 h?P<0.05?,which wereconsistent with the transcriptome sequencing.3.Western blot confirmed that the expression of TRIM22,14,21,25 were significantly increased at 36 h and 48 h after RSV infection.4.Immunofluorescence staining confirmed that the expression of TRIM22 was significantly higher in the A549 cells infected with RSV than in the control group.TRIM22 localized in the cytoplasm with a diffuse and/or a speckled pattern.Summary:1.RSV infection induced a variety of TRIM protein changes in A549 cells.2.The expression of TRIM22 increased most significantly in RSVinfected A549 cells.3.TRIM22 in RSV-infected A549 cells localized in the cytoplasm with a diffuse and/or a speckled pattern.Part two Effect of TRIM22 on proliferation of RSV in A549 cellsObjective: To understand the effect of the corresponding TRIM proteins on RSV proliferation in respiratory epithelial cells infected with RSV by knocking down TRIM 22,14,21,25 and TRIM38 with siRNA.Select TRIM22 for further test through knockdown and overexpression experiments to clarify the role of TRIM22 on the proliferation of RSV in respiratory epithelial cells.Methods:1 TRIM proteins 22,14,21,25 and TRIM38 were knocked down by siRNA,and RT-qPCR was used to verify the knockdown effect of TRIMs in A549 cells.2 After TRIM22,14,21,25 and TRIM38 were knocked down in A549 cells,A549 cells were infected with RSV with MOI=5 for 24 h.RT-qPCR was used to detect the effect of knocking down the corresponding TRIM proteins on RSV N gene.3 TRIM22 was knocked down by siRNA in A549 cells,and A549 cells were infected with RSV with MOI=5 for 48 h.Western blotting confirmed theknockdown effect of TRIM22.4 TRIM22 was knocked down by siRNA in A549 cells.A549 cells were infected with RSV with MOI =5 for 24 h.RT-qPCR was used to detect the changes of N,M,NS1,M2,G and F gene expression in RSV.5 The p LV[Exp]-Puro-EF1A>{hTRIM22 [NM₀06074.4]/3xFLAG}eukaryotic expression plasmid was constructed.A549 cells were transfected with the eukaryotic expression plasmid for 72 h.Western blotting verified the overexpression of TRIM22.6 A549 cells overexpressing TRIM22 were infected with RSV with MOI=5 for 48 h.RT-qPCR was used to detect the overexpression of TRIM22 and the expression changes of RSV N,M,NS1,M2,G and F genes.7 Use siRNA to knock down TRIM22 in A549 cells.RSV-mGFP infected A549 cells with MOI=5 for 48 h.Observe the green fluorescence of RSV-mGFP in TRIM22 knockdown A549 cells.8 Use plasmid transfected A549 cells to overexpress TRIM22,RSVmGFP infected A549 cells with MOI=5 for 48 h,observe the proliferation of RSV-mGFP green fluorescence in A549 cells overexpressing TRIM22.Results:1.A549 cells were transfected with the corresponding siRNA for 24 h.RT-qPCR experiments confirmed that siRNA TRIM22,14,21,25 and TRIM38 can effectively knock down the TRIM proteins mRNA expression level of A549 cells?P<0.05?.2.A549 cells knocked down TRIM22,14,21,25 and TRIM38 were infected with RSV for 24 h.RT-qPCR confirmed that RSV N genes are lower after TRIM22,21 and TRIM25 knockdown?P<0.05?.There was no significant change in the expression of RSV N gene after TRIM14,38 knockdown.3.A549 cells knocked down TRIM22 were infected with RSV for 48 h.Western blot confirmed that TRIM22 knockdown reduced the expression of TRIM22 in the TRIM22 group.4.A549 cells knocked down TRIM22 were infected with RSV for 24 h.RT-qPCR experiments confirmed that the expression levels of N,M,NS1,M2,G,and F genes in RSV were significantly lower than those in controls?P<0.05?.5.A549 cells were transfected with pLV[Exp]-Puro-EF1A>{hTRIM22[NM₀06074.4]/3xFLAG} eukaryotic expression plasmid for 72 h.Western blot confirmed that the effect of overexpression of TRIM22 was significant.6.RT-qPCR experiments confirmed that A549 cells over-expressing TRIM22 infected RSV for 48 h.The expression of N,M,NS1,M2,G and F genes in RSV was significantly higher than that in the control group?P<0.05?.7.RSV-mGFP was used to infect A549 cells for 48 h.The green fluorescence of RSV-mGFP was less in A549 cells knocked down TRIM22 than in the control group;the green fluorescence of RSV-mGFP increased in A549 cells over-expressing TRIM22 compared to the control group.Summary:1.The expression level of RSV N gene was decreased after knocking down TRIM22,TRIM21 and TRIM25 in A549 cells.2.TRIM22 promoted the proliferation of RSV in A549 cells.Part three TRIM22 promoted RSV replication by binding autophagy-related proteinsObjective: To search the autophagy of A549 cells induced by RSV,use siRNA to knock down TRIM22,14,21,25 and TRIM38 to study the effects of TRIMs,especially TRIM22,on the autophagy induced by RSV infection,and to explore the possible mechanism of TRIM22 promoting RSV replication.Methods:1 RSV infected A549 cells 0 h,24 h,48 h,total proteins was extracted,and the expression of LC3 II was detected by Western blot.A549 cells were infected with RSV for 48 h,and the expression of LC3 protein was observed under fluorescence microscope.2 The total proteins was extracted from A549 cells infected with RSV at0 h,24 h and 48 h.The expression of ULK1 and Beclin1 was detected by Western blot.3 The A549 cells were infected with RSV for 0 h,24 h and 48 h.Theexpression of autophagic flow marker protein P62 was detected by Western blot.The cells were treated with BafA1?10 nM?for 2 h to inhibit the fusion of autophagosome and lysosome,and then infected with RSV.The cells were detected for 0 h,48 h and Western blot to detect the expression of P62 and LC3 II.4 A549 cells were transfected with siRNA and TRIM22,14,21,25,38 were knocked down.A549 cells were infected with RSV for 24 h.The expression of LC3 II was detected by Western blot.5 TRIM22 was knocked down in A549 cells and infected with RSV for24 h and 48 h.The expression of LC3 II was detected by Western blot.TRIM22 was knocked down and infected with RSV for 48 h in A549 cells.The expression of LC3 was observed by fluorescence microscope.6 A549 cells overexpressing TRIM22 were infected with RSV for 36 h.Negative controls were also set up.Whole cell lysates were coimmunoprecipitated by anti-Flag magnetic beads.Western blot was used to detect the binding of ULK1,Beclin1 and TRIM22,and whole cell lysates were tested.Results:1.RSV infected A549 cells and the expression of LC3 II was detected by Western blot.The results showed that at 24 h and 48 h after RSV infection,LC3 II was significantly higher than the control.A549 cells were infected with RSV for 48 h.Fluorescence microscopy showed that LC3 was scattered in the cytoplasm of A549 cells.2.RSV infected A549 cells 0 h,24 h and 48 h,the expression of ULK1 and Beclin1 were detected by Western blot.The results showed that the expression of ULK1 and Beclin1 was significantly increased at 24 h and 48 h after RSV infection.3.RSV infected A549 cells 0 h,24 h and 48 h,Western blot was used to detect the expression of autophagic flow marker protein P62.The expression of P62 was not significantly changed at 24 h and was significantly decreased at 48 h.The expression of LC3 II and P62 were significantly increased afterapplication of BafA1,which is used o inhibit the fusion of autophagosome and lysosome?10 n M?,suggesting that RSV infection induces a complete autophagic flow response in A549 cells.4.Western blot results showed that after knocking down TRIM22,14,21,the expression of LC3 II induced by RSV was significantly lower than that of control;the expression of LC3 II induced by RSV after knocking down TRIM25 and TRIM38 did not change significantly compared with the control group.5.Western blot results showed that in A549 cells knocked down TRIM22 and infected with RSV 24 h and 48 h,LC3 II expression levels were reduced compared to the control group;Knocking down TRIM22 in A549 cells and infecting A549 cells with RSV for 48 h,the expression of LC3 observed under fluorescence microscope was reduced compared to the control.6.Coimmunoprecipitation confirmed that TRIM22 interacted with autophagy-related proteins ULK1 and Beclin1.Summary:1.RSV infection induced the increase of LC3 II in A549 cells,and LC3 protein was scattered in the cytoplasm.2.RSV infection induced high expression of autophagy-related proteins ULK1 and Beclin1 in A549 cells.3.RSV infection induced a complete autophagic flow response in A549 cells.4.Knockdown of TRIM22 inhibited RSV-induced autophagy.5.TRIM22 enhanced autophagy by binding autophagy-related proteins ULK1,Beclin1 and then promoted RSV replication.Conclusions:1.RSV infection can induce elevated expression of TRIM22,TRIM14,TRIM21,TRIM25 and TRIM38 in A549 cells.2.Knockdown of TRIM22 inhibited the proliferation of RSV in A549 cells.Overexpression of TRIM22 promoted the proliferation of RSV.3.TRIM22 promoted RSV replication by acting as platforms assemblingautophagy-related proteins ULK1 and Beclin 1.