Study On Signaling Functions,Inter-Relationship And Antiviral Activities Of Porcine RIG-I-Like Receptors RIG-I,MDA5 And LGP2

Innate immunity plays a crtical role in the host defense against the invasion of pathogenic microorganisms.Innate immune receptor family RIG-I like receiptors(RLRs)play an important role in the process of recognizing virus infection,inducing host interferon response and fighting against virus.RLRs include RIG-I,MDA5 and LGP2.Upon recognizing the pathogen associated molecular patterns(PAMPs),RIG-I and MDA5 will be activated,interact with signal adaptor mitochondria antiviral signaling proteins(MAYS)through the homologuous CARD domains,and finally activate transcription factors IRF3 and NF-κB,inducing the expression of type I interferom(IFN)and inflammatory cytokines.Subsequently,interferon signaling cascade will be activated to fight viral infection.LGP2 has no signal active domain CARDs,without signaling activity itself,but mainly exerts its function by regulating the activity of RIG-I and MDA5.Despite the extensive studies on human and mice RLRs,currently,there is a lack of systemic investigation of livestock animal RLRs.Therefore,we isolated the porcine RIG-I,MDA5 and LGP2,and investigated their signaling functions,mutual relationship and antiviral effects.1.Functional analysis and antiviral effects of porcine RIG-I-like receptors RIG-I,MDA5 and LGP2Porcine RIG-I,MDA5 and LGP2 gene codings were isolated from porcine aveolar macrophages(PAMs),inserted into pENTR4-MCS vector,and the porcine RIG-I,MDA5 and LGP2 were transferred into pDEST-cDNA vectors by LR site specific recombination reaction,to obtain pRIG-I,pMDA5 and pLGP2 eukaryotic expression plasmids,respectively.The expressed protein sizes of pRIG-I,pMDA5 and pLGP2 were 120 kDa,140 kDa and 80 kDa,respectively,in transfected cells examined by Western-blotting.In transfected HEK 293T cells,both pRIG-I and pMDA5 showed constitutive activity and pMDA5 showed stronger signaling activity,whereas pLGP2 has no activity.In response to different RNA ligands and various virus infections,pRIG-I and pMDA5 showed similar signaling activity indicating the similar recognition of different agonists by these two receptors.CRISPR/Cas9 method was used to make porcine RIG-I,MDA5 and LGP2 knockdown PAM and PK15 cells.In pRIG-I and pMDA5 knockdown PAMs/PK15 cells,the transcription levels of downstream genes IFNβ,ISG56,ISG60 and IL8 induced by low molecular weight poly I:C stimulation,VSV or EMCV infection were significantly reduced compared with those in control cells.Although pLGP2 has no signaling activity,the transcription levels of downstream genes in pLGP2 knockdown PAMs/PK15 cells were also decreased.To further ensure the functions of porcine RLRs,pRIG-/-,pMDA5-/-and pLGP2-/-PAMs were prepared by limited dilution and gene sequencing.In these PAMs,the transcriptional levels of IFNβ,ISG56,IL-β,IL8 and TNFα induced by low or high molecular weight poly I:C were remarkably decreased;the replications and gene expressions of VSV,EMCV,SeV,H9N2 and HSV1 were increased and the transcription levels of downstream IFNβ,ISG56,IL1β,IL8 and TNFa genes decreased significantly;additionally,the EMCV induced phosphorylation of downstream transcription factor IRF3 and the protein expression of ISG56 were also notably downregulated.These results suggest that:ⅰ pRIG-I and pMDA5 are able to sense and recognize different RNA and viruses to trigger similar cell signaing,which is different from human and mice RIG-I/MDA5;ⅱ pRIG-I,pMDA5 and pLGP2 might have broad spectrum antiviral functions;ⅲ pLGP2 might have similar regulation of pRIG-I and pMDA5 signaling.Porcine RIG-I,MDA5 and LGP2 were ectopically expressed in PAMs and Marc-145 cells,respectively,and the expressed receptors were all showed to significantly suppress viral replications and gene expressions of swine influenza virus H9N2 and porcine reproductive and respiratory syndrome virus JXA1-R.On the other hand,the ectopical expression of pRIG-I and pLGP2 in IPEC-J2 cells also inhibited the replication of porcine epidemic diarrhea virus YC2014.These results suggested that porcine RLRs play an important role in the resistance to various porcine epidemic virus infections.2.Positive regulation of porcine RIG and MDA5 signaling activites by porcine LGP2Porcine LGP2 has no signaling activity,but we found that in transfected PAMs,pLGP2 could promote the transcriptions of IFNβ,ISG60,IL8 and TNFα genes in a concentration-dependent manner in response to low molecular weight poly I:C stimulation or VSV infection,suggesting the positive regulations of porcine RIG-I/MDA5 signaling by LGP2.Correspondingly,the reductions of RNA and virus induced downstream gene expressions in pLGP2 knockdown PAMs and PK15,as well as pLGP2-/-PAMs also implicated the positive regulation of porcine RIG-I/MDA5 signaling activity by pLGP2.To dessect the respective regulation of porcine RIG-I and MDA5 by pLGP2,the pLGP2 was transfected into pMDA5-/-and pRIG-/-PAMs,respectively,and the results showed that VSV induced IFNβ and ISG56 transcriptons were both increased.It suggested that pLGP2 positively regulated pRIG-I signaing as well as pMDA5 signaling.Mechanically,the results of laser confocal microscopy and immunoprecipitation assay showed that LGP2 could interact with RIG-I and MDA5 after cell activation.By prokaryotic expression and protein purification,we purified pRIG-I,pMDA5 and pLGP2 proteins.RNA binding assay using the purified proteins showed that pLGP2 could promote the binding of pRIG-I and pMDA5 with RNA ligand poly I:C in vitro.In order to explore the responsible sites of pLGP2 on regulating pRIG-I and pMDA5,we constructed porcine LGP2 domains,domain deletion mutants,and point mutants with defects in ATPase and/or dsRNA binding.Intriguingly,we found that most porcine LGP2 mutants showed constitutive activities,especially the domains L1,L2,and point mutants MIIa and MⅢ showing strong constitutive activities.Accordingly,confocal laser microscopy showed that domains L1,L2 and MIIa mutant exhibited obvious nuclear localization.When all the pLGP2 mutants were expressed in pRIG-/-and pMDA5-/-PAMs,respectively,the constitutive activities of most pLGP2 mutants were generally decreased relative to those in control PAMs,suggesting the involvement of RIG-I and MDA5 in the pLGP2 mutant constitutive activities.The reconstitution of pLGP2 and MIII mutant in pLGP2-/-PAMs rescued both antiviral activity and virus induced IFNβ and ISG56 gene transcriptions.3.Porcine RIG-I and MDA5 signaling CARD domains exert similar antiviral function in spite of distinct signaling activityAlthough RIG-I and MDA5 have similar molecular structures,these two receptors have distinct features during activation.Further,the signaling domains of the N terminal CARD domains(CARDs)in RIG-I and MDA5 share poor similarity in amino acid sequences,which was less than 20%.Therefore,we wondered if the CARDs of RIG-I and MDA5 play similar roles in signaling and antiviral function.We isolated and expressed the CARDs of porcine RIG-I and MDA5,and both protein sizes were about 30 kDa,with the molecular weight of MDA5 CARDs obviously higher than RIG-I CARDs.Considering that both CARDs are 200 amino acids,there might be different post-translational modifications in RIG-I and MDA5 CARDs.The CARDs of porcine RIG-I and MDA5 were transfected into PAMs,respectively,and the constitutive activities of CARDs of porcine RIG-I and MDA5 was observed,which were stronger than the corresponding the full-length proteins.The results of laser confocal microscopy showed that both types of CARDs had nuclear localization in contrast to the cytoplasmic localization of full-length proteins,which might be one of the reasons for the higher constitutive activities of CARDs.The signaling activity of pMDA5 CARDs was significantly higher than that of pRIG-I CARDs,which was consistent with the result that the signaling activity of full-length pMDA5 was higher than that of pRIG-I.Nevertheless,the two types of CARDs showed comparable broad range of antiviral activities.The pRIG-I and pMDA5 CARDs in transfected PAMs not only significantly inhibited the RNA viruses such as VSV,EMCV,SeV,influenza viruses H9N2 and H1N1,but also protected host against the infection of DNA virus HSV-1.Transcriptome sequencing showed that MDA5 CARDs had more effective activity in regulating downstream genes.Ectopic expression of MDA5 CARDs in PAMs resulted in 511 up-regulated and 240 down-regulated downstream genes,while RIG-I CARDs induced only 266 up-regulated and 33 down-regulated genes.This result was consistent with the higher signaling activity of MDA5 CARDs.However,in the presence of H9N2 infection,two types of CARDs induced similar number of downstream up-regulated and down-regulated genes.The results suggested that porcine RIG-I and MDA5 CARDs exhibit both disparity and similarity in signalling activity and antiviral function.