Proteomic Analysis Of DTMUV-infected Cells And The Effects Of DDX3X On DTMUV Replication

Duck Tembusu virus disease is a newly emerging disease in duck flocks caused by duck Tembusu virus(DTMUV).DTMUV infection firstly causes a decline of feed intake in duck flocks,and a decrease in egg production,which drops to 10% in 10 d,will promptly develop.The mortality ranges from 5% to 30%.DTMUV is a positive-sense single-stranded RNA virus,belonging to genus Flavivirus,family Flaviviridae.Not only can the DTMUV affect the productivity in laying ducks and breed ducks,but it also infects meat-type ducks,geese,chickens,sparrows,wild birds,mosquitos and even mice.DTMUV disease is an infectious disease,which has uncertain transmission routes and wide host range and spreads rapidly.Currently,no effective medicines against DTMUV disease are available.Therefore,this disease causes huge economic losses to the waterfowl industry and poses a great threat to the public health.In this study,iTRAQ approach was employed to quantitatively identify differentially expressed cellular proteins in DTMUV-infected BHK-21 cells for the first time.Further,we also analyzed the role of DDX3 X in DTMUV replication in host cells.This study provides theoretical direction for prevention and control of the disease.The detailed research contents are as follows:1.Proteomic analysis of DTMUV-infected BHK-21 cells To explore the protein change of DTMUV-infected BHK-21 cells,iTRAQ approach combined with 2D-LC–MS/MS analysis was used to identify the differentially expressed proteins after DTMUV infection.A total of 192 proteins were identified to be differently expressed,among which 11 significantly upregulated and 8 downregulated at 24 hours post infection(hpi).At 48 hpi there were 25 upregulated proteins and 151 downregulated.These proteins were analyzed by bioinformatic analysis,including GO,KEGG pathway and protein interaction networks,which showed that differentially expressed proteins were involved in several biological processes,cell components and molecular functions.The differentially expressed proteins at 48 hpi were far more than those at 24 hpi.TLR9,the most significantly upregulated protein,and other two representative proteins(DDX3X,DDX5)at 48 hpi were selected for confirmation by qRT-PCR and western blot analysis.The results keep in agreement with those identified with iTRAQ approaches,suggesting TLR9,DDX3 X and DDX5 may affect DTMUV replication.2.Research on the effects of DDX3 X on DTMUV replication To study the role of DDX3 X in DTMUV replication,DDX3 X was cloned from BHK-21 cells and the eukaryotic expression plasmid 3Flag-DDX3 X was successfully constructed.And the virus titer was detected after overexpression or knockdown of DDX3 X in BHK-21 cells as well as infection with DTMUV.The results showed that the overexpression of DDX3 X decreased virus replication and knockdown of DDX3 X promoted virus replication.It has been reported that DDX3 X has different functions in the life cycles of different viruses and inhibits dengue virus in human embryonic kidney(HEK293T)cells by regulating interferon(IFN)pathway through TBK1.In order to determine whether DDX3 X has a similar effect in BHK-21 cells,we infected BHK-21 cells with VSV-GFP which is sensitive to IFN,and found the replication of VSV-GFP is inhibited after poly(I:C)stimulation,suggesting that the IFN pathway exists in BHK-21 cells.We immediately compared the amino acid sequence of DDX3 X between human and hamster and found the similarity index as high as 98.3%.HEK293 T cells were transfected with eukaryotic expression plasmids of 3Flag-DDX3 X and HA-TBK1.Then,the immunoprecipitation experiments were performed,which suggested that HA-TBK1 interacted with 3Flag-DDX3 X.The activity of IFN-? promoter was measured by dual luciferase reporter system,and the results showed that the hamster-derived DDX3 X significantly promoted the activity of the IFN-? promoter compared to the control or single-transfected HA-TBK1 experimental group,indicating that the hamster-derived DDX3 X was involved in the induction of TBK1-mediated IFN-? promoter.Meanwhile,the total RNA of HEK293 T cells was extracted with Trizol Reagent and qRT-PCR was used to detect the mRNA levels of IFN-? and downstream genes.The results showed that co-transfection of 3Flag-DDX3 X and HA-TBK1 significantly promoted mRNA transcription of IFN-? and downstream genes including STAT1,OAS,Mx1,ISG15 and ISG56.It is further demonstrated that hamster-derived DDX3 X has similar functions to human DDX3 X,which can enhance the IFN-? expression through TBK1.