The Roles Of LGP2 Protein In The Zebrafish ZF4 Cells Infected With Snakehead Fish Vesiculovirus

Retinoic acid inducible gene I-like receptors(RLRs) are the important members of pattern-recognition receptors(PRRs). RLRs can recognize intracellular viral RNAs,subsequently activate downstream interferon expression pathway. Therefore, they play a critical role in innate immune responses against virus infection. Up to date, there are three members of RLRs, named Retinoic acid inducible gene-I(RIG-I), Melanoma differentiation-associated gene 5(MDA5) and Laboratory of genetics and physiology 2(LGP2). In this study, two LGP2 isforms(Dr LGP2 a and Dr LGP2b) were cloned from zebrafish, and the expression profiles of RLRs and related genes were assessed in zebrafish embryonic fibroblast cells(ZF4) during the infection of SHVV. The results were as follows:1. The full-length c DNAs of Dr LGP2 a and Dr LGP2 b were sequenced. The c DNA sequences of Dr LGP2 a and Dr LGP2 b were deposited in NCBI, and the accessed number was KP341002 and KP341003, respectively. The full-length c DNA of Dr LGP2 a was2473 bp which consisted of a 5’ terminal untranslated region(UTR) of 50 bp, a 3’ UTR of383 bp, and an open reading frame(ORF) of 2040 bp encoding a polypeptide of 579 amino acids. The predicted amino acid sequences showed that Dr LGP2 a contained an ATP dependent DEXDc domain, an RNA helicase HELICc domain(HELICc) and a regulatory domin(RD). Dr LGP2 b was a splicing isoform of Dr LGP2 a. They both shared DEXDc and HELICc domains, but Dr LGP2 b was lack of RD domain.2. ZF4 cells were infected with SHVV and the infection features of SHVV were investigated. The expressions of m RNA and c RNA of viral N and G genes were significantly up-regulated at 3 h post of infection(poi). However, they were dramatically decreased during 6-24 h poi; The amount of IFN-α m RNA was significantly up-regulated at 6 h poi and reached the peak at 12 h poi. The amount of interferon inducing gene Mx m RNA was significantly up-regulated at 6 h poi and reached the peak at 24 h poi; The amounts of m RNAs of RIG-I, MDA5 and LGP2 genes were all significantly increased at 3 h poi, and reached to the peak at 6 h poi.. Taken together, RLRs mediated IFN expression pathway might be activated in ZF4 cell during the infection of SHVV. To confirm that the RLRs-IFN pathway was indeed induced by the SHVV infection, ZF4 cells were transfected with total cellular RNAs extracted from ZF4 cells infected with or without SHVV. The results showed that only total RNAs from ZF4 cells infected with SHVV at 3 h poi were able to significantly up-regulate the expression of IFN-α m RNA,indicating that the viral replicative intermediates may involve in the activation of RLRs-IFN pathway.3. The generated polyclonal antibody of Dr LGP2-DEXDc was used to assess the expression of the two LGP2 isoforms in ZF4 cells during the infection of SHVV. The amount of Dr LGP2 a protein was about ten times of Dr LGP2 b protein. Significant over expression of Dr LGP2 a protein was detected at 12 h poi, indicating that the two isoforms may play different roles in the regulation of RLRs-IFN pathway.