Abstract

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 ( As ISG15) was cloned and characterized from the marine fish black seabream, Acanthopagrus schlegelii . The full-length cDNA of As ISG15 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 As ISG15 shared 31–70% amino acid identity with other known ISG15s and had a closer evolutionary relationship with teleost ISG15s. In vitro , As ISG15 expression was inducible by poly I:C, LPS and red spotted nervous necrosis virus (RGNNV) in cultured black seabream brain cells. In vivo , As ISG15 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 As ISG15 significantly increased RGNNV replication in RGNNV infected AsS cells, and the LRGG domain was crucial for the anti-RGNNV activity of As ISG15. By promoter-driven luciferase reporter assay, we demonstrated that two IFN-stimulated response elements within the promoter region of As ISG15 and the promoter-proximal intron were essential for As ISG15 expression. Furthermore, our results showed that the gamma-IFN activation sequence located in the intron was required for the intron mediated enhancement for As ISG15 expression. Our results would provide insights for understanding the underlying regulation mechanism of ISG15 in teleost.