Study Of BAG5 From Orange-spotted Grouper (Epinephelus Coioides) Involved In Nervous Necrosis Virus Infection

BAG(Bcl-2-associated athanogene)protein family is a kind of Bcl-2-binding proteins that play important roles in cell death regulation and immune response against pathogen infection.BAG5 is a member of this family.However,little is known about the functions of fish BAG5.In this study,we characterized a BAG5 homolog from orange-spotted grouper(Epinephelus coioides)gene(Ec-BAG5)for expression analysis and its role during viral infection was investigated by transfection in vitro,quantitative Real-time PCR and luciferase assay.The main results are as follows:1.The ORF of Ec-BAG5 was 1407 bp,encoding a polypeptide of 468 amino acids with a calculated molecular mass of 53.2k Da and a p I of 5.93.The Ec-BAG5 protein contains four conserved BAG family domains,phylogenetic analysis showed that it had the closest relationship with Large yellow croaker(Larimichthys crocea).2.The m RNA expression of Ec-BAG5 could be tested in various tissues,the highest transcriptional level of Ec-BAG5 was found in the peripheral blood lymphocyte(PBL),and the lowest was in spleen.And the Ec-BAG5 expression were significantly up-regulated in grouper spleen(GS)cell after red-spotted grouper nervous necrosis virus(RGNNV)or Lipopolysaccharide(LPS)stimulation.Subcellular localization analysis showed that the protein encoded by BAG5 was distributed in the whole cell.3.Ec-BAG5 overexpression could inhibited viral replication and the expression of viral genes(coat protein(CP)and RNA dependent RNA polymerase(Rd Rp)).Further analysis showed that overexpression of Ec-BAG5 significantly increased the expression of interferon pathway-related factors including interferon regulatory factor3(IRF3),interferon-stimulated gene 15(ISG15),interferon-induced protein 35(IFP35),myxovirus resistance gene 1(Mx1)and inflammatory-related factors including tumor necrosis factor receptor-associated factor 6(TRAF6),tumor necrosis factor-α(TNF-α),interleukin-1 beta(IL-1β).Moreover,the overexpression of Ec-BAG5 significantly increased NF-κB,ISRE and IFN-1 promoter activities.