Signaling And Anti-viral Functions Of Chicken Innate Immune DNA Sensoring CGAS-STING Axis

Innate immune system detects foreign pathogen aassociated molecular patterns(PAMPs)by pattern recognition receptors(PRRs).Upon recognition of DNAs,the DNA sensors recruit adaptor MyD88 or STING to induce downstream type I interferons(IFNs),inflammatory cytokines and other gene expression products to fight the infections.cGAS is a novel cytoplasmic DNA sensor discovered in 2013,it can be activated by pathogenic double-stranded DNAs and then transmit signaling to downstream adaptor protein STING via synthesized second messenger 2’-3’-cGAMP to induce the expression of type I IFNs and other related genes.cGAS-STING pathway not only plays an important role in anti-DNA virus infection immunity,but also be closely related to tumors and autoimmune diseases.Therefore,our study investigates chicken cGAS and STING focusing on their signaling activity,mutual relationship and anti-viral functions.1.Gene cloning,signaling functions and tissue distributions of chcGAS and chSTING chcGAS and chSTING were amplified from chicken HD11 cells by RT-PCR using specific cloning primers which designed according to the reference sequences of chcGAS and chSTING in GenBank.The gene fragments were inserted into pENTER4-MCS-3FLAG vectors,and the chcGAS and chSTING were transferrred into pDEST vectors by LR site specific recombination reaction,to obtain chcGAS and chSTING eukaryotic expression plasmiids,respectively.The protein sizes of chcGAS and chSTING were 60 kDa and 45 kDa,respectively,in transfected cells examined by Western-Blotting.Co-transfection of chcGAS and chSTING in 293T cells could not activate ISRE promotor,instead,significantly activated the activity of NF-κB promoter and downstream hIL-1β,hIL-8.By contrast,co-infection of porcine cGAS and STING in 293T cells could activate ISRE promotor;besides,co-infection of chcGAS and pSTING could also activate ISRE promoter in 293T cells.The results suggested that cGAS and STING have species-specificity between mammal and poultry,and STING plays a key role in the species-specificity.Tranfection of chcGAS or chSTING alone in chicken HD 11 cells was sufficient to activate chIFN-β promoter and downstream chIFN-β chIL-1β,chIL-8,and the activity was further significantly increased by both co-transinfection.The above experiments showed that both chcGAS and chSTING are signaling functional and exert their roles via cGAS-STING axis.The tissues of three-week-old SPF chicken were collected to detect the mRNA distributions of chcGAS and chSTING.The results showed that chcGAS mRNA was the highest in the pancreatic gland,while chSTING mRNA in the spleen,and both were generally higher in the intestine.2.Study on the interaction between chcGAS and chSTING and the signaling active sites of chcGASCo-IP experiments showed that chcGAS and chSTING could co-precipitate in the form of immune complex,indicating that there was an interaction between chcGAS and chSTING proteins.In this study,the potential magnesium,zinc,and ATP/GTP binding sites of chcGAS were analyzed by mutation assay for their roles in signaling activity,which include S129,E141,D143,D239,E303,N309,H310,C317,C324,K334 and S353-K357.The results from HD 11 transfection showed that all chcGAS mutants could not activate the downstream chIFN-β promotor and type I IFN induction,Among different mutants,S129A,D239A,H310A still kept the inducing activity of downstream chIL-1β and chIL-8,while others could not activate chIL-1β and chIL-8.The results from 293T transfection showed that mutants E141A and N309A had normal NF-κB promoter activity and hIL-1β,hIL-8 induction;however,other mutants lack the NF-κB promoter activity and hIL-1β,hIL-8 induction.The discrepancy of results from transfected 293T and HD11 cells,once again,proved the species-specificity between mammals and poultry.3.Study on anti-viral functions of chcGAS and chSTINGIn this study,chcGAS and chSTING were expressed in HD 11 cells by lentivirus infection,respectively,and then the stable expression HD 11 cells were obtained by puromycin selection.Simutaneously,chcGAS and chSTING CRISPR gRNAs were expressed in HD 11 cells by lentivirus infection,respectively,and then chcGAS KO and chSTING KO stable cells were obtained from puromycin selection.The characterizations of stable expression and KO HD cells were achieved by stimulations with 45bp-dsDNA,pcDNA3.1,and HSV-1,respectively.Stimulation of the chcGAS and chSTING stable expression HD 11 led to higher expression of downstream chIFN-β,chOASL,chIL-1β and chIL-8 compared with those from control HD11 cells.Meanwhile,chcGAS and chSTING KO stable HD11 cells showed reduced response to stimulations relative to control CRISPR HD 11 cells.Stable expression and KO HD 11 cells were used to examine the roles of chcGAS and chSTING in anti-viral immunity.Poxvirus VACV strain,Vesicular stomatitis virus(VSV),Sendai virus(SeV),Encephalomyocarditis virus(EMCV),and Avian leukemia virus(ALV)all activated downstream both IFN and NF-κB pathways;while Poxvirus SMV strain and Newcastle disease virus(NDV)mainly activated downstream NF-κB pathway.The replication levels of VACV,SMV,VSV,SeV,NDV and ALV were inhibited in chcGAS and chSTING stable expression HD11 cells;whereas the replication levels of these viruses increased in chcGAS and chSTING stable KO HD11 cells.The above used viruses contain DNA viruses,RNA viruses and retrovirus,thus the results demonstrate that chcGAS-chSTING signal axis have broad-spectrum anti-viral function.