Identification of two ISG15 homologues involved in host immune response against RGNNV in Asian seabass (Lates calcarifer).

Interferon Stimulated Gene (ISG)15 is a ubiquitin-like protein that is induced upon viral infections. Our study reports the identification of two homologues of ISG15 in the Asian seabass designated LcISG15A and LcISG15B. The cloned LcISG15A cDNA fragment contained a 474 bp ORF encoding a 157 amino acid protein whereas LcISG15B featured a 498 bp ORF encoding a slightly longer protein of 165 amino acids. Both proteins featured the two tandem ubiquitin-like domains and the C-terminal LRGG motif characteristic of ISG15. The LcISG15B protein has a 10-amino acid C-terminal extension after the LRGG motif. Molecular docking studies revealed that LcISG15A showed more conformational variability of the ubiquitin domains and catalytic function than LcISG15B. The Lates ISG15A and ISG15B genes, reside close in the genome, share the same basic structure with two exons and an intron, but only the second exon encoding the protein. These genes also featured the IFN-stimulatory response elements (ISRE) in the promoter region and ATTTA instability motif in the 3' UTR region. Leukocyte-rich organs such as the head kidney, heart, spleen, and gill showed higher levels of ISG15A and ISG15B basal expression. Poly (I:C) injection rapidly upregulated the transcription of both the ISG15 genes in these tissues in Lates. In-vivo viral infection by red-spotted grouper nervous necrosis virus also induced upregulation of ISG15 genes in the head kidney, spleen, heart and gill. These findings indicate that the two ISG15 homologues may play a crucial role in innate antiviral immunity and could be used to improve prophylactic strategies and develop species-specific immunological tools for Lates calcarifer.