Novel Findings in Teleost TRAF4, a Protein Acts as an Enhancer in TRIF and TRAF6 Mediated Antiviral and Inflammatory Signaling.

Tumor necrosis factor receptor-associated factors (TRAFs) are important adaptor molecules that play important roles in host immune regulation and inflammatory responses. Compared to other members of TRAFs, the function of TRAF4 in vertebrate immunity remains unclear, especially in teleosts. In the present study, TRAF4 ortholog was cloned and identified in large yellow croaker (), named as . The open reading frame (ORF) of - is 1,413 bp and encodes a protein of 470 amino acids (aa), which is consisted of a RING finger domain, two zinc finger domains, and a MATH domain. The genome organization of - is conserved in teleosts, amphibians, birds, and mammals, with 7 exons and 6 introns. Quantitative real-time PCR analysis revealed that - was broadly distributed in various organs/tissues of healthy large yellow croakers and could be significantly up-regulated in the gill, intestine, spleen, head kidney, and blood under poly I:C, LPS, PGN, and stimulations. Notably, luciferase assays showed that overexpression of -TRAF4 could significantly induce the activation of IRF3, IRF7, and type I IFN promoters, with the RING finger and zinc finger domains function importantly in such promoter activation. Confocal microscopy revealed that -TRAF4 is located in the cytoplasm, whereas the deletion of the RING finger, zinc finger or MATH domain showed little effect on the subcellular localization of -TRAF4. Interestingly, -TRAF4 overexpression could significantly enhance -TRIF and -TRAF6 medicated IRF3 and IRF7 promoter activation. In addition, co-expression of -TRAF4 with -TRIF or -TRAF6 could significantly induce the expression of antiviral and inflammation-related genes, including , , , , , , , and compared to the only overexpression of -TRAF4, -TRIF or -TRAF6. These results collectively imply that -TRAF4 functions as an enhancer in -TRIF and -TRAF6 mediated antiviral and inflammatory signaling.