Study On Chicken RIG-I-like Receptor MDA5 And Downstream Linker Protein MAVS

Innate immunity is the body’s first defense barrier.A series of pattern recognition receptors(PRRs)encoded by innate immune genes recognize pathogen-associated molecular patterns(PAMPs)produced by various pathogenic microorganisms.The main receptor family that recognizes RNAs are cytosolic RIG-I like receptors(RLRs),including RIG-I,MDA5 and LGP2.MDA5 can recognize long double-stranded RNA and pass signals to the downstream adaptor protein MAVS to mediate a complex network of signal pathways,produce interferons,pro-inflammatory factors and related chemokines,and finally initiate innate immunity against pathogenic microorganisms.Most reports of MDA5 showed that it recognizes RNA viruses,but some studies have also demonstrated that MDA5 can recognize RNAs produced by DNA viruses and parasites during replication.Chicken MDA5(chMDA5)plays a key role because chickens lack RIG-I.In this study,chicken-derived MDA5 was cloned from chicken cells,chMDA5 and chMAVS eukaryotic expression plasmids were constructed,and the expression and function of chMDA5 and chMAVS were verified.The antiviral effects of chMDA5 and chMAVS and their signaling relationship were examined by the overexpression and CRISPR knockout system.Finally,the chMDA5 prokaryotic expression plasmid was constructed,soluble chMDA5 was purified from bacteria and polyclonal antibodies were prepared from the immunized mice to provide services for subsequent experiments.1.Cloning,expression and function of chMDA5 and chMAVSIn this study,chMDA5 was cloned from HD11 cells and ligated into the pENTER4 intermediate vector to obtain the recombinant intermediate plasmid pENTER4-chMDA5-3FLAG.The synthesized chMAVS was similarly ligated into pENTR4 vector to obtain recombinant pENTR4-chMAVS-3FLAG.The positive intermediate plasmids were subjected to LR reactions with the destination vector pDSET47 to obtain the recombinant eukaryotic expression plasmids pcDNA-chMDA5-3FLAG and pcDNA-chMAVS-3FLAG,respectively.The correct expressions of chMDA5 and chMAVS in transfected 293T cells were confirmed by Western blotting.Fluorescence confocal microscopy showed that both chMDA5 and chMAVS were both localized in the cytoplasm,but there was no obvious co-localization.The signal functions of chMDA5 and chMAVS were detected by the dual luciferase reporter gene system and RT-PCR.Both chMDA5 and chMAVS activated the NF-κB pathway in co-transfected of 293T cells,while co-transfection activated the IFNβ pathway in DF1 cells.RT-PCR showed that co-transfection in 293T cells up-regulated the transcription of IL-1 and IL-8,whereas co-transfection in DF-1 cells upregulated IFN and OASL.The above-mentioned differences suggested the cell species specificity.In addition,the signal adaptor protein also exhibits species specificity.Compared with the aforementioned chMDA5 and chMAVS co-transfection of 293T cells,where only NF-κB is activated,co-transfection of chMDA5 and porcine(p)MyD88 in 293T cells can effectively activate both ISRE and NF-κB signals.The qRT-PCR method was applied to detect the distributions of chMDA5 and chMAVS mRNAs in different tissues of chickens.It was found that the distributions of chMDA5 and chMAVS were relatively similar,mainly in the digestive glands and some intestines,followed by lungs and spleen.2.Effects of chMDA5 and chMAVS overexpression and knockout on virus replicationsDF1 cells were used to transfect chMDA5 and chMAVS expression plasmids alone or in combination.The transfected cells were stimulated by infections with different viruses,including RNA virus Vesicular Stomatitis Virus(VSV),Sendai Virus(SeV),Encephalomyocarditis Virus(EMCV),chicken Newcastle Disease Virus(NDV)and DNA viruses(Vaccinia strains VacV and SMV).qRT-PCR was used to detect the expressions of viral replication genes and downstream genes in infected cells.According to the known sequences,the gRNA coding DNA sequences(primers)of chMDA5 and chMAVS were designed by using Benchling software respectively.The annealed primers were ligated to the plentiCRISPRv2 vector to obtain recombinant knockout plasmids gMDA5 and gMAVS.Knock-out gRNA plasmids and corresponding eukaryotic expression plasmid were co-transfected into 293T cells to test gRNA knock-out efficiency by Western-blotting,and the gRNA plasmid with better knock-out effect was selected for cell transfection experiment.DF1 cells were transfected with gMDA5 and gMAVS gene knockout plasmids alone or in combination.Transfected cells were also stimulated by infections with the above-mentioned different RNA and DNA viruses.qRT-PCR was used to detect the expression of virus replication genes and downstream genes in infected cells.The results showed that in the transfected expression and knockout cell system,chMDA5-chMAVS could respond to RNA viruses VSV,SeV,EMCV,NDV and produce IFN and OASL to influence virus replications.In the DF-1 cells expressing chMDA5 and chMAVS,the above RNA virus replications were all inhibited,while the RNA virus replications in the knockout DF-1 cells were all enhanced.At the same time,the co-expression of chMDA5 and chMAVS could further inhibit RNA virus replications,while in the co-expressoin of gMDA5 and gMAVS could further enhance the the replications of the RNA viruses.Therefore,chMDA5 and chMAVS play a crucial role in the innate immunity against RNA viruses.The results of DNA virus infections showed that endogenous chMDA5 and chMAVS did not affect the virus replications and expressions of downstream virus stimulating genes,so chMDA5-chMAVS had no significant effect in restricting DNA virus infections.3.chMDA5 polyclonal antibody preparationThe recombinant prokaryotic expression plasmid was obtained by LR recombination between pENTER4-chMDA5-3FLAG and the prokaryotic destination vector pDSET527.The recombinant expression plasmid was transformed into E.coli BL21(DE3)and the bacteria were induced with IPTG to express the His-chMDA5-3FLAG protein.The soluble prokaryotic chMDA5 proteins with the expected size were purified and concentrated from the supernatant of the lyzed bacteria.Mice were immunized with the purified protein 15 μg per mouse each time,and serum was collected after three immunizations.Through Western blot detection,it was found that the serum polyclonal antibodies could detect exogenous MDA5.