The Identification Of Chicken MDA5-STING-IFN-β Anti-viral Pathway And Its Signal Transduction

Innate immunity is the first line of host defense against invading pathogens.Innate immune responses are initiated by the host’s pattern recognition receptors(PRRs),which recognize conserved pathogen-associated molecular pat-terns(PAMPs)of microorganisms.After recognition of the PAMPs,the PRRs trigger signals that induce the IFNs and the inflammatory cytokines that suppress the replication of invading pathogens.The RIG-I like receptors(RLRs)are cytosolic pattern recognition receptors of viral RNA.RLRs are the most important PRRs in response to RNA virus infection.After be activated by RNA virus,the RLRs trigger signals resulting the the expression of type I IFN which suppress the replication of invading viruses.The mammalian RLR family consists of three members: retinoic acid-inducible gene I(RIG-I),melanoma differentiation-associated gene 5(MDA5)and laboratory of genetics and physiology 2(LGP2).RIG-I plays the most important role in anti-RNA virus infections among the three members of RLRs.Although the RLR pathway is generally conserved among vertebrates,and the RIG-I has been identified in duck,goose and pigeon,however,it was found that RIG-I is missing in chicken cells,putting the function of chicken MDA5(chMDA5)under the spotlight.Though there are initial studies on chicken MDA5(chMDA5),the related researches are less detailed.The signal pathway mediated by chMDA5 to induce IFNs in chicken cells and its role in anti-virus are not clear.Stimulator of IFN genes(STING)is an important adaptor involed in the innate immunity.STING senses the RNA virus and indhces the type Ⅰinterferons through the RIG-I-STING-IRF3-IFN-β signal transduction axis.Interestingly,STING was proved not involved in the MDA5 mediated type Ⅰ interferon pathway.genome.A single STING-like chicken gene(chSTING)was found on chickne chromosome 13.So we are wondering if the mammalian STING-like chicken gene chSTING has the same function as the mammalian STING in anti-RNA virus? We are also interested in the relationships between the chSTING and chicken RLRs.The discovery of chSTING gene makes the mechanisms of chMDA5 mediated type Ⅰ interferon pathway more confusing.In this study,We identified chicken STING(chSTING)as a critical mediator of virus-triggered type I IFN signaling in RIG-I–null chicken cells.We investigated the molecular basis of chSTING for its biological functions.More importantly,we identified a new type Ⅰ interferon pathway,mediated by chMDA5 and relying on chSTING,which is very different from mammalian cells.We firstly cloned the immune factors that might involved in the chMDA5 pathway,such as,chMDA5,chMAVS and chIRF7.The overexpression and knockdown experiments showed that all the three genes were all IFN related genes,and they played a role in anti-H9N2 AIV.This study laid the foundations for the further and integral studies of chMDA5 pathway.Though RIG-I gene is missing,a STING-like gene was identified in chicken cells.We clonded chSTING for the first time and found chSTING shared a low similarity(43.3%)with its counterparts in human.Tissue and cell distribution study showed that chSTING mRNA could be detected in all the tissues tested and with highest level in immune organs.chSTING predominantly localized to the outer membrane of the endoplasmic reticulum and was also found in the mitochondrial membrane.The chSTING overexpression and knockdown experiments confirmed its function in IFN induction and its role in anti-RNA and-DNA virus.This study cloned the chSTING gene and identified it as an essential gene in IFN induction and anti-RNA virus.This study determined the functional domains and the key amino acids of chSTING through the functional domains deletion and site-directed mutagenesis experiments.The result showed that all the four TM domains of chSTING are indispensable for its subcellular localization and IFN induction.Deleted any one of the TM domains decreased the IFN production.The site-directed mutagenesis experiments indicated that post-translational modification,such as phosphorylation and ubiquitination,might be involed in chSTING signal transduction.This stdy indentified the S366 as an important amino acid site for chSITNG as its mutation made chSTING defective in IFN induction.Several lines of evidence suggest that STING is involved only in RIG-I signaling,but not MDA5 signaling,in mammalian cells.Knockdown of chSTING significantly blocked the IFN-β production induced by both poly(I:C)and the overexpression of chMDA5-N,and this indicated that chSTING might participate in the chMDA5 pathway.Co-immunoprecipitation(Co-IP)experiments found that chSTING was constitutively associated with chMDA5 and chMAVS,and this confirmed that chSTING participated in the chMDA5 pathway in RIG-I–free chicken cells.The further study showed that the presence chMAVS in the Co-IP system could enhance the interaction between chMDA5 and chSTING.Domain-mapping experiments indicated that the chMDA5 CARD domain,CARD+DEXD domain and the MDA5 full lenth colud interact with chSTING to trigger the immune response.The CARD truncation mutant of chMDA5 could not activate the IFN-β pathway,however,it could still interact with chSTING.In mammalian cells,STING activate IFN-β through Interferon regulatory factor-3(IRF-3).However,IRF3 is also missing in chicken cells.With the software analysis we found three IRF cis-acting elements in chicken IFN-β promoter.Overexpression of chSTING and chIRF7 could enhance the activities of IRFs and IFN-β promoter drived luciferase report gene,while knockdown of chSITNG has a opposite effect.This indicated that chSTING might activate the IFN-β through chIRF7,which was IRF3 in mammalian cells.We then conducted the overexpression and gene-specific knockdown experiment.The result demonstrated the chMDA5-chMAVS-chSTING-chIRF7-IFN-β signal transduction axis in chicken cells,in which chSTING was functioning downstream of chMAVS and upstream of chIRF7.In this study,we identified a new type Ⅰ interferon pathway(chMDA5-chMAVS-chSTING-chIRF7-IFN-β)in RIG-I missing chicken cells.In this pathway,chSTING receives the activation signal trigerred by chMDA5 through the upstream chMAVS,and chSTING activates the IFN-β through the downstream IRF7.We also provided evidences to confirm that the chMDA5 mediated and chSTING relied IFN pathway play an important role in anti-RNA virus.The newly discovered pathway in chicken cells breaks the traditional theory that MDA5 could not induce IFNs through STING.This study also demonstrated that chSTING activates the IFN-β through chIRF7 pathway,even if IRF3 is also missing in chicken cells.We speculate that the newly discovered pathway might be a compensation mechanism to compensate for the gene defects in chicken cells: The missing of RIG-I and IRF3 break the RIG-I-STING-IFN-β pathway,however,chMDA5 could compensate for the lack of RIG-I by interacting with chSTING to construct the MDA5–STING pathway in chicken cells.At the same time,chIRF7 could work as IRF3 to compensate for the lack of IRF3 to reconstruct IFN pathway.