DDX1 From Cherry Valley Duck Mediates Signaling Pathways And Anti?NDRV Activity

When the host is infected by pathogenic microorganisms,the innate immune system recognizes pathogen-associated molecular patterns(PAMPs)through the pathogen molecular pattern recognition receptors(PRRs),and activates downstream signaling pathways to induce host production effector molecules and inflammation.The helicase protein,as a cytoplasmic nucleic acid recognizer,plays an important role in innate immunity.DEAD-box RNA helicases constitute the largest type of nucleic acid helicases and are a highly conserved family of RNA binding proteins.They are named because they contain a unique Asp-Glu-Ala-Asp(DEAD)sequence motif,except for it can act as an adenosine triphosphate(ATP)-dependent RNA helicase to promote changes in RNA structure in various cell activities,and can also be used as a sensor for the occurrence of metabolic diseases.DEAD-box helicase 1(DDX1)is a member of the DEAD-box RNA helicase family,and it was first discovered in retinoblastoma and neuroblastoma in 1993.It can recognize double-stranded RNA(ds RNA)viruses,and can affect the antiviral immune response by regulating the expression of interferons(IFNs)in the host’s innate immune response.Novel duck reovirus(NDRV),as a new infectious disease,is a member of the orthoreovirus and belongs to the ds RNA virus.Its pathological changes are manifested by hemorrhage and necrosis in the liver and spleen.The disease has a high incidence and a wide host range,and it will die due to secondary infections.The lack of effective prevention methods will cause serious economic losses to the waterfowl breeding industry.DDX1 plays a critical role in the innate immune system against viral infections,but the role of duck DDX1(duDDX1)in the anti-NDRV infection has not yet been elucidated.This study was the first to study the biological function of DDX1 in Cherry valley duck and its role in the innate immune response of anti-NDRV.The main findings are as follows:Part Ⅰ Cloning,identification,and biological analysis of DDX1 gene of Cherry valley duckAccording to the predicted primers on NCBI,RNA was extracted from the spleen of healthy Cherry valley duck and reverse transcribed into cDNA.The full-length coding sequence(CDs)of duDDX1 was cloned.The full length of duDDX1 consists of 2223 bp and encoded740 amino acids with a molecular weight of 82 k Da.The eukaryotic expression plasmid pcDNA3.0-duDDX1-Flag was constructed.Through biological analysis,we found that duDDX1 was composed of three domains: DEXDc domain,HELICc domain,and SPRY domain,and has the highest homology with chicken DDX1.It can be seen that it has the closest relationship with Anas platyrhynchos on the phylogenetic tree.Part Ⅱ Distribution of DDX1 in Cherry valley duckRNA was extracted from heart,liver,spleen,lung,kidney,brain,cerebellum,muscle,cecum,ileum,jejunum,duodenum,trachea,bursa of fabricius,muscle stomach,glandular stomach,skin,esophagus,pancreas,and brain stem to determine the distribution of duDDX1 in various tissues.Quantitative real-time PCR(q RT-PCR)found that duDDX1 is widely distributed in all tissues tested,especially in the duodenum,liver,and spleen.The experimental results of infecting ducks with NDRV showed that the expression of duDDX1 was significantly up-regulated in the liver,but significantly down-regulated in the spleen,this suggests that the specific response of duDDX1 is different in the two organs infected with NDRV,and the host may participate in the antiviral immune response mainly by up-regulating the expression of duDDX1 in the liver.Part Ⅲ duDDX1 participates in duck’s innate immune response by mediating IFN-β related signaling pathwaysOverexpression of duDDX1 in DEF cells,through q RT-PCR detection,it was found that the m RNA expression levels of Toll-like receptors(TLR2,TLR3,and TLR4)were significantly up-regulated,but the expression levels of MDA5 and RIG-I were not significantly changed.In addition,the expression levels of OAS,IL-8,IL-1β,and IFNs were significantly up-regulated.The dual luciferase experiment found that the transcriptional activity of the IFN-β promoter was significantly activated,and the transcription factors IRF-7 and NF-κB were activated at the same time,which proved that both the IRF-7 and NF-κB promoters are involved in the regulation of duDDX1 induced activation of IFN-β signaling pathway.After overexpressing duDDX1 and infecting NDRV in DEF cells,the expression of TLR3 was down-regulated,but the expressions of TLR2 and TLR4 were up-regulated,and there was no significant change in MDA5 and RIG-I.The expression of other cytokines such as OAS,Mx,and IL-6 was significantly down-regulated.In the reverse experiment of knocking down the expression of duDDX1,it can be seen that duDDX1 regulates the expression of some PRRs,cytokines,and antiviral proteins.For example,at 36 hpi,IFN-β was up-regulated by 2.1 times(P <0.001),IL-6 was down-regulated by 2.4 times(P <0.001),and IL-8 was down-regulated by 2.0 times(P<0.001).This indicated that duDDX1 may participate in the innate immune process of duck by mediating IFN-β related signaling pathways.Part Ⅳ duDDX1 has anti-NDRV activityDEF cells were transfected with pcDNA3.0-duDDX1-Flag or empty vector,and infected with NDRV.We found that overexpression of duDDX1 can significantly inhibit the replication of NDRV in the early stage,and the above conclusions were further verified by interference experiments on DEF cells.Experiments show that,overexpression of duDDX1 will inhibit the replication of NDRV,and duDDX1 has anti-NDRV activity.