Sequence structure, tissue expression of LGP2, and its interaction with MDA5 in Squaliobarbus curriculus

Grass carp (Ctenopharyngodon idella) are susceptible to grass carp reovirus (GCRV) and exhibit severe hemorrhage when infected. In contrast, Squaliobarbus curriculus (Sc) are resistant to GCRV. We have generated C. idella/S. curriculus hybrid fish which are relatively more resistant to GCRV than C. idella. These fish populations provide valuable resources for investigating the genetic mechanisms of the antiviral immunity. The laboratory of genetics and physiology 2 (LGP2) is a member of RIG-Ⅰ like receptor family and is involved in recognition of viral nuclear acid at the first line of defense. Understanding its functions in antiviral immunity will have a significant impact on the molecular breeding of fish with resistant traits. In this study, the full-length cDNA (2 940 bp) and the 5′ upstream sequence (721 bp) of the S. curriculus lgp2 (Sclgp2) gene were sequenced. The 5' upstream region contains several putative transcription factor binding sites for MafB (muscle aponeurosis fibromatosis B) and IRF3 (interferon regulatory factor 3). The Sclgp2 encodes a protein of 680 amino acids which consists of conserved DEXD/H (DExD/H-box helicase domain), HELICc (helicase superfamily C-terminal domain) and CTD (C-terminal regulatory domain). The functional domains and the number of predicted phosphorylation sites are similar to that in other fish species, with differences in domain arrangement and amino acid sequences. Furthermore, by comparing the sequences of C. idella and S. curriculus LGP2 proteins, two GCRV sites associated with GCRV resistance in the RNA binding region were identified. In the phylogenetic tree, ScLGP2 was clustered in a clade containing the homologs from C. idella, Carassius auratus and Mylopharyngodon piceus, forming an expanded clade with the homologs from other cyprinid fish. Quantitative PCR analysis revealed that the mRNA expression level of Sclgp2 was the highest in the spleen, followed by muscle and heart, whereas the lowest was detected in the intestine. The expression levels of Sclgp2 and ifn1 remained unchanged in most tissues except that the ifn1 expression was significantly down-regulated in the liver at 24-72 h after GCRV infection. Interestingly, the mRNA expression levels of Sclgp2 in the muscle positively correlated with that of ifn1 (0.999, P<0.01). ScLGP2 weakly interacted with ScMDA5, yet DEXDc (1-201 aa), HELICc (390-476 aa) and CTD (553-668 aa) were not engaged in such interaction. Taken together, the present study provides basis for studying the functions of ScLGP2 and exploring the Sclgp2 gene as a potential marker for genetic breeding of disease resistant C. idella.