Study On The Effect And Mechanism Of Duck DEAD-box RNA Helicase 47 And 3 On Duck Tembusu Virus Replication

Duck Tembusu virus(DTMUV)first broke out in China in 2010,which mainly caused laying duck severe ovarian hemorrhage and acute egg-drop syndrome,and seriously endangered the healthy development of the Chinese duck industry.DTMUV belongs to the genus flavivirus of the family Flaviridae,and it is the first flavivirus to cause serious disease in waterfowl so far.In-depth study of the interaction between DTMUV and host can provide a better understanding of the flavivirus immunologic and pathogenesis mechanism,and have important public health significance.In this study,an IFN-β dual luciferase reporting system was built to study duck innate immunity.The epidemiological DTMUV strain isolated in our laboratory was used as the research strain,the differentially expressed genes of primary duck embryo fibroblasts(DEFs)infected with DTMUV were analyzed by transcriptome sequencing(RNA-Seq),and screened a series of effector molecules related to TMUV-induced host innate immune.The molecular structure,expression characteristics and biological functions of duck DEAD box RNA helicase 47(du DDX47)and 3(du DDX3)were analyzed.Moreover,the mechanism of du DDX47 promoting DTMUV proliferation was discussed in this study.The main contents were as following:1.Construction of the dual luciferase reporter system of duck IFN-β promoter and its IRF1 and NF-κB binding sitesIn order to investigate the relationship between DTMUV infection and host innate immune response,the putative promoter fragment of IFN-β gene was cloned from duck spleen c DNA by PCR based on the reported duck interferon beta promoter sequence.The Luciferase reporter vector pdu IFN-β-Luc containing duck IFN-β gene core promoter region and the Luciferase reporter vector 4×IRF1-Luc and 4×NF-κB binding site containing four repeated sequences of IRF1 and NF-κB were constructed respectively.The constructed reporter vectors were transfected into DEFs respectively,detected after 24 hours of stimulation with poly(I:C).The results showed that the three dual luciferase reporter vectors were constructed successfully.2.Transcriptomic analysis of DTMUV-infected host cellsRNA-Seq was performed on primary DEF cells infected with virus for 24 h and uninfected cells,and data was uploaded to National Center for Biotechnology Information(NCBI)Sequence Read Archive(SRA)(NCBI-SRA Accession No:SUB7261213).Totally,1838 differentially regulated genes were identified,including649 upregulated and 1189 downregulated genes.The Gene Ontology(GO)functional classification and KEGG signaling pathway enrichment of 1838 differentially expressed genes screened by RNA-seq showed that DTMUV infection were related to the innate immune signaling pathway.It mainly includes pattern recognition receptor signaling pathways(RIG-I,NOD-like and Toll-like)related to activate IFN-β signaling pathway,NF-kappa B signaling pathway related to inflammatory response,and C-type lectin receptor signaling pathway related to virus invasion lectin receptors signaling pathway.The significantly differentially expressed genes,IFN-a、IFN-β、viperin、Mx1、IRF1、IFIT5 and OASL,were selected for q RT-PCR validation,the trend was consistent with those identified with RNA-Seq approach.According to the information provided by RNA-Seq,DDX47 and DDX3 that significantly upregulated genes were selected for further study.3.Research on the mechanism of du DDX47 promoting DTMUV proliferationThe c DNA of duck DDX47 was firstly cloned from duck spleen c DNA by based on the predicted du DDX47 sequence.The complete CDS of the du DDX47 gene deposited in NCBI Gen Bank(Accession No: OQ626368).was 1389 bp in length,encoding 463 amino acids,contains a DEAD domain(aa172-675)and a HELICc(aa859-1104).Du DDX47 was expressed in multiple duck tissues,with the highest m RNA expression in the spleen and pancreas.Subcellular localization analysis showed that du DDX7 was mainly located in the nucleus,especially in the nuclear membrane.It was verified that the overexpression of du DDX47 promoted DTMUV replication and knockdown of du DDX47 decreased DTMUV replication by q RT-PCR and IF detection.Further analysis of du DDX47 expression in primary DEF cells infected with TMUV demonstrated that TMUV up-regulated DDX47 transcription and protein expression in a dose-dependent manner.To comprehensively understand which TMUV-encoded proteins could interact with du DDX47 and affect its expression,eukaryotic expression plasmids of all ten TMUV encoded proteins were constructed.By using co-transfection and coimmunoprecipitation(Co-IP)the interaction was detected,three structural protein(Caspid、Pr M、Envelope)and four non-structural proteins(NS1、NS2A、NS4A、NS4B)encoded by TMUV were determined to be interacted du DDX47.Considered that du DDX47 belongs to RNA helicase,which could interact with viruses through RNA,ribonuclease A was added to the cell lysate for Co-IP assay.The results showed that du DDX47 could directly interact with Caspid,Pr M,NS1,NS4 A and NS4 B,independent of RNA.Consistent results were obtained by reverse Co-IP,indicating that the interaction between du DDX47 and DTMUV Caspid,Pr M,NS1,NS4 A and NS4 B is a direct interaction independent of RNA.Further indirect immunofluorescence and laser confocal analysis also confirmed the colocalization of du DDX47 with seven proteins encoded by DTMUV in the cytoplasm.Western Blot analysis showed that du DDX47 significantly up-regulated the expression of Caspid and NS4 A in a dosedependent manner.Caspid and NS4 A were not only related to DTMUV immune escape,but also closely related to virion assembly and endoplasmic reticulum reorganization induced by flavivirus replication.Therefore,we speculated that du DDX47 plays an important role in the replication of DTMUV.We deduced that du DDX47 could decrease IFN-β expression because du DDX47 promotes remarkably DTMUV proliferation.it was expectedly found that overexpression of du DDX47 significantly inhibited the duck IFN-β promoter activity in a dose-dependent manner regardless of background expression or Poly(I:C)stimulation,and overexpression of du DDX47 inhibited IFN-β production by inhibited the transcription factors IRF1 and NF-κB dependent promoters.Further truncated mutants based on du DDX47 predicted domains were constructed,our results indicated that the du DDX47 aa254-463 region containing the HELICc and C-terminal domains seems essential to inhibit IFN-β promoter in DEF cells.Meanwhile knockdown of du DDX47 in DEFs by si RNA significantly promoted poly(I:C)-and DTMUV-induced IFN-β activation.Therefore,du DDX47 also promoted DTMUV replication by inhibiting IFN-β expression.4.Molecular cloning and function analysis of du DDX3It has been reported that du DDX3 has different antiviral effects on flavivirus members,promoting WNV replication but inhibiting DENV replication.However,the function of duck DDX3 X has not been reported.The primers of DDX3 X were designed based on the predicted sequence from NCBI.The du DDX3 was obtained from the duck spleen total RNA through reverse transcription method.Full-length du DDX3 c DNA were 1959 bp encoded 652 amino acid(aa)residues containing nine signature motifs,a DEADc domain and a HELICc domain.The phylogenetic analysis based on du DDX3 aa sequences indicated that du DDX3 was grouped into the aves clade and the highest sequence similarity with chicken DDX3 X.According to the tissue distribution analysis,du DDX3 m RNA was widely expressed in different tissue,especially the spleen and liver.Subcellular localization of du DDX3 was detected,it was found that du DDX3 was distributed throughout the cytoplasm and nuclear in DEFs.When analyzing the anti-TMUV effect of du DDX3 by q RT-PCR and Western Blot,which found that overexpression of du DDX3 had some effect on DTMUV replication,but the difference was not significant compared with the control.Overexpression of du DDX3 could activate NF-κB and IRF1 to induce IFN-β production by dual luciferase reporter assay,and knockdown of endogenous du DDX3 expression could significantly reduce the poly(I:C)-induced IFN-β promoter activation.To identify the key domain of du DDX3 in inducing IFN-I production,eukaryotic expression plasmids expressing four truncated du DDX3 mutants were constructed.By dual-luciferase reporter assay,it were confirmed that truncated mutants of du DDX3(165-400)and du DDX3(1-400)that lacking HELICc domain and containing HELICc domain were unable to activate the IFN-β promoter.This study indicated that HELICc is the key domain of du DDX3 to induce IFN-β production.