Expression pattern, antiviral role and regulation analysis of interferon-stimulated gene 15 in black seabream, Acanthopagrus schlegelii

Interferon stimulated gene 15 (ISG15) is an IFN inducible ubiquitin-like protein and plays a critical role in immune response against viral infection. In this study, an ISG15 gene (AsISG15) was cloned and characterized from the marine fish black seabream, Acanthopagrus schlegelii. The full-length cDNA of AsISG15 was 1302 bp and encoded 155 amino acids containing two ubiquitin-like motifs and a LRGG conjugation domain. Multiple alignment and phylogenetic tree showed that AsISG15 shared 31-70% amino acid identity with other known ISG15s and had a closer evolutionary relationship with teleost ISG15s. In vitro, AsISG15 expression was inducible by poly I:C, LPS and red spotted nervous necrosis virus (RGNNV) in cultured black seabream brain cells. In vivo, AsISG15 was ubiquitously expressed in all examined tissues with higher expression levels in eye and gill, and the expression was significantly up-regulated in most tissues post RGNNV infection, especially in liver, spleen and kidney. The testing of antiviral activity showed that silencing AsISG15 significantly increased RGNNV replication in RGNNV infected AsS cells, and the LRGG domain was crucial for the anti-RGNNV activity of AsISG15. By promoter-driven luciferase reporter assay, we demonstrated that two IFN-stimulated response elements within the promoter region of AsISG15 and the promoter-proximal intron were essential for AsISG15 expression. Furthermore, our results showed that the gamma-IFN activation sequence located in the intron was required for the intron mediated enhancement for AsISG15 expression. Our results would provide insights for understanding the underlying regulation mechanism of ISG15 in teleost.