MDA5 and LGP2 acts as a key regulator though activating NF-κB and IRF3 in RLRs signaling of mandarinfish

Abstract

RIG-I-like receptors (RLRs), as key cytoplasmic sensors of viral pathogen-associated molecular patterns, can recognise viral RNA and enhance the antiviral response. Some investigations have focused on the roles of RLRs in the innate immune response in grass carp, large yellow croaker, and rainbow trout. However, little is known about the function of RLRs in mandarinfish (Siniperca chuatsi), an important economic fish in Perciformes. Here, we functionally characterized the RLRs involved in the immune responses of mandarinfish (Siniperca chuatsi), by evaluating three RLRs, namely, RIG-I, MDA5, and LGP2. The results revealed that MDA5 and LGP2 were present in mandarinfish, whereas RIG-I was absent. The MDA5 and LGP2 cDNA sequences contained 2976 and 2046 bp and encoded 991 and 681 amino acids, respectively. Multiple sequence alignments showed that MDA5 and LGP2 of mandarinfish were clustered together with their homologs from other teleost fishes and shared high similarities with those from other vertebrates, and RIG-I of mandarinfish was absent. Moreover, quantitative real-time PCR (qPCR) analysis suggested that MDA5 and LGP2 were constitutively expressed in all tissues tested, and MDA5 mRNA expression was relatively high in the gill, and spleen, whereas LGP2 mRNA expression was high in the liver, gill, and head kidney. After stimulation with lipopolysaccharide or poly I:C, the expression of MDA5 and LGP2 was upregulated in spleen, gill and head kidney, but the pattern was not exactly the same, MDA5 transcripts generally increased and then declined with the prolonged infection, while LGP2 transcripts went up continuously, which showed that mandarinfish MDA5 and LGP2 may play independent roles in antiviral response. Besides, it is further revealed that the MDA5 could activate NF-κB and IRF3 to inducing the production of IFN-β by constructing tet-on stable strain of 293T cell, however over-expression of LGP2 resulted in decreased NF-κB, IRF3 and IFN-β production in cells challenged with LPS and polyI:C Taken together, our results demonstrated that MDA5 and LGP2, as a positive and negative regulator, respectively, played an important role in modulating antibacterial andantiviral immune responses though activating NF-κB and IRF3 in RLRs signaling of mandarinfish.

Keywords: Innate antiviral immunity; Lipopolysaccharide; Mandarinfish; Poly I:C; RIG-I-like receptor.