Screening Of Proteins Interacted With Chicken MDA5 And Transcriptome Sequencing Analysis Of Chicken Mda5 Overexpressed DF1 Cells

As the first line of defense for the body to resist the invasion of external pathogenic microorganisms,innate immune plays an important role in protecting the body’s health.It mainly relies on pattern recognition receptors(PRRs)to recognize pathogen-associated molecular patterns(PAMPS)of pathogenic microorganisms to induce the production of interferon,thereby exerting antiviral effects and protecting the health of the body.The RIG-I-like receptor(RLR)family is an important member of cytoplasmic PPRs.And retinoic acid-inducible gene I(RIG-I),melanoma differentiation-associated gene 5(MDA5)and laboratory of genetics and physiology 2(LGP2)are three members in the RLR family.MDA5 and RIG-I can recognize different types of viral RNA and exert antiviral effects.While,LGP2 plays a role in regulating the activities of RIG-I and MDA5 due to the lack of CARD region.Different from mammals,RIG-I is absent in chickens naturally.Thus,the role of MDA5 in anti-viral natural immunity is extremely important for chickens.However,unlike the deep and extensive studies on mammalian MDA5,the regulation of chicken MDA5-mediated signaling pathway remains unclear.In this study,by the methods of mass spectrometry and next-generation sequencing,we explored the mechanisms of chicken MDA5 signaling regulation.The main results are as follows: 1.Identification of proteins that interacted with chicken MDA5 and the study on the mechanism of these proteins in regulating chicken IFN-β production induced by chicken MDA5.By the way of GST-pull down combined with mass spectrometry,we identified 336 host factors that could potentially interact with chicken MDA5.Though GO and KEGG analysis,we found that these host factors are mainly involved in heterocyclic compound binding,protein binding,and ion binding in molecular biological functions,etc.Interaction network analysis revealed that these host factors are mainly involved in the lysosomal,the spliceosome,RNA transport and protein processing,etc.Then we cloned 60 genes and studied the effects of these 60 genes on the chicken IFN-β promoter activity.After that we chose 10 genes that can affect the promoter activity of chicken IFN-β under the stimulation of both chicken MDA5-N and chicken MDA5(chicken DDX5,EEF1A1,RBM39,PABPC1,HSPA5,HSPA8,HSP70,hn RNPH2,IFT5,PRDX1)and explored influences of these genes on the production of chicken IFN-β.Next,through CO-IP and IFA experiments,we found that the six proteins,containing DDX5,HSPA8,HSP70,hn RNPH2,IFIT5,PRDX1,could interact with chicken MDA5-N.Further studies on chicken hn RNPH2 suggested that chicken hn RNPH2 could inhibit chicken IFN-β production,most likely because it could impair the interaction between chicken MDA5 and chicken MAVS.2.Transcriptome sequencing analysis of DF1 cells overexpressing chicken MDA5 and DF1 cells stimulated by double-stranded RNA mimic poly(I:C)The bioinformatics analysis was performed on the sequencing data to screen differentially expressed genes.It was found that in the chicken MDA5 group,there were 1,007 differentially expressed genes,containing 737 up-regulated genes and 270 down-regulated genes.While,in the poly(I:C)group,there were 745 differentially expressed genes.Among them,405 genes were up-regulated and 340 genes were down-regulated.Moreover,85 genes were differentially expressed in both the chicken MDA5 group and the poly(I:C)group,including 62 up-regulated genes and 23 down-regulated genes.The GO and KEGG analysis of differentially expressed genes manifested that the top 20 pathways in the chicken MDA5 group mainly involved to immune-related pathways and that in the poly(I:C)group were related to biosynthesis and metabolism and influenza virus infection.Next,we selected 9 genes containing chicken MX1,IFI6,IFIT5,RSAD2,OASL,CMPK2,HELZ2,EPSTI1 and OLFML1,which were up-regulated in both the chicken MDA5 group and the poly(I:C)group,to verify the reliability of the transcriptome data by real time PCR.Further study found that chicken MX1,IFI6,IFIT5,RSAD2,and OASL could inhibit the proliferation of influenza virus H5N6 in DF1 cells.Our study not only deepened the understanding of the mechanism of chicken MDA5 signaling regulation but also laid a theoretical foundation for chicken antiviral research.