Cloning And Fuctional Analysis Of Differentiation-associated Gene 5(MDA5) Promoter And Preliminary Application

The innate immune system acts as the first defensive barrier against invading pathogens,using pattern recognition receptors(PRRs)to recognize viral pathogen-associated molecular patterns(PAMPs).PRRs consists of several receptor families,such as toll-like receptors(TLRs),nucleotide-binding oligomerization domain protein-like receptors(NLRs)and retinoicacid-inducible gene I(RIG-I)-like receptors(RLRs).The RLRs family is composed of RIG-I,melanoma differentiation-associated gene 5(MDA5)and laboratory of genetics and physiology 2(LGP2),all of which play important roles in recognizing viral RNA in the cytoplasm and triggering a series of initial antiviral activities.In mammals,both of RIG-I and MDA5 recognize distinct,but overlapping,families of RNA viruses.RIG-I primarily recognizes viruses that contain varied length RNA with 5’-triphosphates,5’-diphosphates or short double-stranded RNA(dsRNA)that are<1 kb and RNA with specific secondary structures.MDA5,however,chiefly detects long polyinosinic-polycytidylic acid(poly(I:C))and picomaviruses with long dsRNA(>1 kb).In chickens,RIG-I gene is absent and this may provide a rationale for increasing susceptibility to certain pathogens in comparison to ducks and mammals which could express RIG-I.MDA5 is hypothesized to be important in detecting viral nucleic acids in the cytoplasm.Hitherto,the molecular mechanism of chicken MDA5(chMDA5)expression regulation has yet to be fully elucidated.In the current study,we cloned and analyzed the full length promoter of chMDA5 and then explore its reponse to viral and non-viral stimuli,which would lay foundation of breeding for disease resistance poultry.The findings are as following:Firstly,a～2.5 kb full length chMDA5 gene promoter region was amplified by PCR using White Leghorn chicken genomic DNA as the template.Molecular analysis reveals that the chMDA5 promoter contains several putative transcription factor-binding sites,including binding sites for Sp1,GATA-1,TGGCA-binding protein,AP-1,AP-3,ER-alpha,T3R-alpha,C/EBP alpha,NF-1(-like proteins)and NF-E4.In order to.exploit the evolutionary relationship of promoters among chicken and other birds,fish and mammals,a phylogenetic analysis was performed.The chMDA5 promoter demonstrated the closest evolutionary distance to bird MDA5 promoters and the furthest from mammals.Secondly,a chMDA5 promoter reporter plasmid(piggybac-MDA5-DsRed)was constructed and transfected into DF-1 cells to establish a Piggybac-MDA5-DsRed cell line.For the Piggybac-MDA5-DsRed cells,the proportion of DsRed expressing cells represents the promoter activity of chMDA5.FACS analysis and fluorescence microscope observations demonstrated that virtually no DsRed expression could be noted,which meant that the the MDA5 promoter activity was extremely low under basal conditions.However,when the cells were treated with long poly(I:C)and short poly(I:C),interferon beta(IFN-β)or IBDV,the proportion of DsRed-expressing cells transfected with long poly(I:C)and short poly(I:C)were 18%and 9.86%,2.01%and 1.01%,respectively.This meant the activity of chMDA5 promoter was dramatically increased.As the DsRed mRNA level represented the promoter activity,QPCR results showed that the DsRed mRNA level was remarkably upregulated by poly(I:C),IFN-P or IBDV,which matched the expression of endogenous MDA5 and IFN-β.What’more,the long poly(I:C)resulted more DsRed-expressing cells and more fold changs in DsRed and MDA5 mRNA level than short poly(I:C),suggesting that MDA5 has a preference to long poly(I:C).As long poly(I:C)upregulated both IFN-α and IFN-β while short poly(I:C)only upregulated IFN-β,maybe the signaling pathways triggered by long poly(I:C)and short poly(I:C)were not exactly the same.Finaly,a duck RIG-I vector PB-chMDA5-duRIG-I-mkate plasmid was constructed which utilizing chMDA5 promoter to promote the expression of duck RIG-I on PB system.It was demonstrated that the duck RIG-I gene in the PB-chMDA5-duRIG-I-mkate plasmid was able to express in chicken cells when the cells were stimulated by long poly(I:C)while it was unable to be found to express under base conditions.In summary,the results of the current study indicated that the promoter and the Piggybac-MDA5-DsRed cell line could be utilized to determine whether a ligand could regulate MDA5 expression.Importantly,the chMDA5 promoter could be used to drive the expression of other antiviral genes,as its activity is extremely low under basal conditions and could be modulated by exogenous inducers.To our knowledge,this study is the first to clone the promoter of chMDA5 and examine its function.Our results provide a foundation for future research on the direct regulation of MDA5 expression,which could contribute in developing new strategies against viral infection.