| Retinoic acid-inducible gene-I-like receptor（RLR）is an important class of pattern recognition receptors（PRR）.Three members of RLR family have been identified in mammalian,namely retinoic acid induced gene-I（RIG-I）,melanoma differentiation associated gene 5（MDA5）and genetic and physiological experimental gene 2（LGP2）.At present,it is believed that LGP2 mainly plays an auxiliary regulatory role in the signal transduction of RLRs,but it is still far from a thorough understanding of its mechanism.Although the existence of RLRs in ducks has been confirmed,and some studies have also proved that duck RIG-I（duRIG-I）and duMDA5 are involved in the host’s innate immune response to duck Tembus virus（DTMUV）.However,there are few studies on the regulatory role of duLGP2 in the antiviral innate immune response mediated by RLRs.In this study,DTMUV was used as a model virus,and the results will help to further understand the pathogenic mechanism of the disease and develop new antiviral drugs or vaccine adjuvants provide a rationale.In this study,duLGP2 was combined with full-length duRIG-I or duMDA5 and the caspase activation and recruitment domain（CARD）of duRIG-I or duMDA5,respectively,in duck embryo fibroblast（DEF）cells.After co-expression,the regulatory effect of duLGP2 on duRIG-I or duMDA5-mediated signaling pathway was detected by dual-luciferase reporter gene and q RT-PCR.The DEF cells co-expressing duLGP2 with duRIG-I or duMDA5 were further infected by DTMUV,and the differences in the induced immune response and virus replication in the above cells were compared.The main findings are as follows:1.Cloning,biological analysis and subcellular localization of duLGP2 geneAccording to the duLGP2 gene sequence predicted by the NCBI database,amplification primers were designed from both sides of the gene coding region,and the full-length coding region sequence of duLGP2 was amplified from the c DNA of the spleen of the cherry valley duck by PCR.Sequence analysis showed that duLGP2 is 2,028 bp in length,encoding 675 amino acids,and the protein size is about 77.5 k Da.duLGP2 consists of three domains:DEx D/H-box,HELICc and CTD.The eukaryotic expression plasmid pduLGP2-Flag was constructed by ligating duLGP2 to the eukaryotic expression vector p CAGGS,and it was verified that it was expressed in DEF cells and was mainly located in the cytoplasm.2.The regulatory effect of duLGP2 on duRIG-I and duMDA5 signaling pathwaysThe constructed full-length duRIG-I and duMDA5 or eukaryotic recombinant expression plasmids containing only the effector domain CARD were co-transfected with duLGP2 eukaryotic expression plasmid or empty vector,firefly luciferase reporter gene plasmid and internal reference control plasmid,respectively.DEF cells.The results showed that duLGP2 inhibited the duRIG-I and duMDA5-mediated IFN-β signaling pathway in DEF cells in a dose-dependent manner.The expression of pro-inflammatory cytokines,type I IFN,and downstream ISGs mediated by full-length duRIG-I and duMDA5 was significantly downregulated by duLGP2 by q RT-PCR.Interestingly,overexpression of duLGP2 enhanced the expression of pro-inflammatory cytokines when duRIG-I and duMDA5 signaling were activated by their CARD domains.3.Regulation of duLGP2 on duRIG-I and duMDA5 signaling pathways during DTMUV infectionThe present study demonstrates that duLGP2 downregulates duRIG-I and duMDA5-mediated IFN-β signaling during DTMUV infection.q RT-PCR results showed that duLGP2 inhibited duRIG-I and duMDA5-mediated expression of type I IFN and its downstream ISGs during DTMUV infection,and duLGP2 promoted pro-inflammatory responses in duRIG-I and duMDA5-mediated anti-DTMUV immune responses.The expression of cytokines enhanced the inflammatory response mediated by duRIG-I and duMDA5.These results collectively suggest that the anti-DTMUV capacity of duRIG-I and duMDA5 is downregulated by duLGP2,which ultimately leads to enhanced DTMUV replication in DEF cells. |
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