Functional analysis of Epinephelus coioides peroxisome proliferative-activated receptor alpha (PPARalpha): Involvement in response to viral infection.

Peroxisome proliferative-activated receptor alpha (PPARalpha) belongs to the superfamily of nuclear receptors (NR). Studies have demonstrated that PPARalpha functions in energy metabolism, hepatic function, immune response, cell cycle, and apoptosis. In teleost fish, few studies have investigated the role of PPARalpha in the immune response. In this study, the grouper PPARalpha gene (EcPPARalpha) was investigated for its role in viral infection. The open reading frame of EcPPARalpha encoded a protein of 469 amino acids and contained an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region, and a C-terminal ligand-binding domain (LBD). Phylogenetic analysis revealed that EcPPARalpha was most closely related to homologous genes in Sander lucioperca and Perca flavescens. Upon challenge with SGIV (Singapore grouper iridovirus) and RGNNV (Red-spotted grouper nervous necrosis virus), EcPPARalpha expression levels were significantly upregulated in different tissues. Subcellular localization analysis showed that the EcPPARalpha protein localized throughout the cytoplasm and nucleus with diffuse intracellular expression patterns, which is consistent with the localization pattern of mammalian PPARs. Based on morphological observation of cytopathic effect (CPEs), viral gene expression mRNAs, and virus titer assays, the results presented here showed that an overexpression of EcPPARalpha promoted SGIV production in grouper spleen cells. Overexpression of EcPPARalpha significantly inhibited the expression of several cytokines, including interferon-related genes (IFN-gamma, ISG15, MXI, MXII, MAVS and MDA5), inflammatory cytokines (IL-1beta, IL-6, IL-8, TNF-alpha) and Toll like receptor adaptors (TRAF6 and MyD88). Luciferase activity of IFN-alpha, IFN-gamma, ISRE and NF-kappaB promoters was also significantly decreased in EcPPARalpha overexpression cells. Due to these detected interferon-related genes and inflammatory cytokines play important antiviral effect against SGIV in grouper, we speculated that the promotion effect of EcPPARalpha on SGIV replication may be caused by down-regulation of interferon and inflammatory response. In addition, through apoptotic body observation, capspase-3 activity detection, and flow cytometry analysis, it was found that overexpression of EcPPARalpha promoted SGIV-induced apoptosis in fathead minnow (FHM) cells. These data may increase an understanding of the role of PPARalpha in fish antiviral immune responses and apoptosis.