The Study On Relationship Between CGAS-STING Signaling Pathway And MDV Replication

Marek's disease is a malignant infectious tumor caused by T lymphocyte hyperplasia caused by Marek's disease virus(MDV),which causing huge economic losses to the poultry industry.Although the disease has been effectively controlled with the vaccine,it still cannot prevent the spread of MDV.MDV can cause immune suppression and lethal lymphoma in poultry,leading to secondary infections and even death of other viruses and bacteria infection.When MDV establishes an infection in the body,it must first break through innateimmunity.The cGAS-STING signaling pathway can recognize dsDNA,thereby inducing the production of IFN-?.At present,there have been a large number of studies on the herpes virus escape cGAS-STING signaling pathway,but there are few studies on the innate immune escape mechanism of poultry herpes virus.This study focuses on the relationship between cGAS-STING signaling pathway and MDV replication,and in-depth studies on the effects of viral encoded proteins on cGAS-STING signaling pathway,providing a theoretical basis for a further explanation of the immunosuppressive mechanism of MDV.1.Activating cGAS-STING signaling pathway can inhibit MDV replication in CEF cellsIn order to investigate whether the cGAS-STING signaling pathway is contained in poultry cells,the mammalian STING ligand stimulator 2'-3'cGAMP was used to act on chicken embryo fibroblasts(CEF).Fluorescence quantitative PCR detected the upregulation of IFN-? mRNA in CEF cells,suggesting the presence of cGAS-STING pathway in CEF cells.STING ligand stimulator 2'-3' cGAMP can inhibit MDV replication in CEF,indicating that activation of cGAS-STING signaling pathway can reduce MDV replication in CEF;MDV infection of CEF cells found that STING,IRF7 and IFN-? mRNA were all up-regulated,suggesting that MDV can activate the cGAS-STING signaling pathway in CEF cells and promote IFN-? production.2.Preliminary study on cloning expression and biological function of cGAS and STING genes in poultryIn order to further explore the role of cGAS-STING signaling pathway in poultry cells,chcGAS and chSTING genes were amplified by PCR,and homology comparison and phylogenetic tree analysis were performed with mammalian cGAS and STING.The comparison results showed that the homology between chcGAS,chSTING with other birds was about 70%?90%,and the homology between chcGAS,chSTING with mammalian cGAS,STING was about 40%.After tranfection the chcGAS and chSTING gene into cells,the dual-luciferase report experimental results showed that both chcGAS and chSTING can up-regulate the promoter activity of IFN-? and IRF7.Fluorescence quantitative PCR experimental results showed that chcGAS and chSTING could up-regulat the expression of TBK1,IRF7 and IFN-? gene.These results indicate that chcGAS and chSTING play a role in inducing IFN-? in poultry cells.At the same time,overexpression of STING in CEF cells can activate the cGAS-STING signaling pathway and inhibits MDV replication.3.Effect of MDV-encoded protein on cGAS-STING signaling pathwayMDV encoded Meq protein plays an important role in tumor generation,transformation and anti-apoptosis.To establish infection in the body,MDV first needs to break through the innate immunity and escape the host antiviral response.In this study,the meq genes of three MDV strains(RB-1B,AH1410,and CVI988)were cloned and co-transfected with cGAS and STING into CEF cells.It was found that they can inhibit the increase of IFN-? promoter activity caused by cGAS and STING,and this inhibition effect is dose-dependent.Meq was expressed in segments and co-transfected with cGAS and STING in DF-1 cells.The results showed that the amino acid region of Meq exerting its inhibitory effect on IFN-? was located at 271?281aa.These results further validate the immunosuppressive effect of the Meq and provides new targets for the development of new MDV vaccines.