题目: Isolation and characterization of tumor necrosis factor receptorassociated factor 6 (TRAF6) from grouper, Epinephelus tauvina

作者:  Jingguang Wei , Minglan Guo , Pin Gao , Huasong Ji , Pengfei Li , Yang Yan , Qiwei Qin*

刊物：Fish & Shellfish Immunology, (IF=3.034, I区)

刊号:  2014,39:61-68

摘要: Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adapter molecules in Tolllike receptor signal transduction that triggers downstream cascades involved in innate immunity. In the present study, a TRAF6 (named as Et-TRAF6) was identified from the marine fish grouper, Epinephelus tauvina by RACE PCR. The full-length cDNA of Et-TRAF6 comprised 1949 bp with a 1713 bp open reading frame (ORF) that encodes a putative protein of 570 amino acids. Similar to most TRAF6s, Et-TRAF6 includes one N-terminal RING domain (78aae116aa), two zinc fingers of TRAF-type (159aae210aa and 212aae269aa), one coiled-coil region (370aae394aa), and one conserved C-terminal meprin and TRAF homology (MATH) domain (401aae526aa). Quantitative real-time PCR analysis revealed that Et-TRAF6 mRNA is expressed in all tested tissues, with the predominant expression in the stomach and intestine. The expression of Et-TRAF6 was up-regulated in the liver after challenge with Lipoteichoic acid (LTA), Peptidoglycan (PGN), Zymosan, polyinosineepolycytidylic acid [Poly(I:C)] and Polydeoxyadenylic acid$Polythymidylic acid sodium salt [Poly(dA:dT)]. The expression of Et-TRAF6 was also up-regulated in the liver after infection with Vibrio alginolyticus, Singapore grouper iridovirus (SGIV) and grouper nervous necrosis virus (GNNV). Recombinant Et-TRAF6 (rEt-TRAF6) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-Et-TRAF6 serum preparation. Intracellular localization revealed that Et-TRAF6 is distributed in both cytoplasm and nucleus, and predominantly in the cytoplasm. These results together indicated that Et-TRAF6 might be involved in immune responses toward bacterial and virus challenges.

题目: Involvement of the PI3K and ERK signaling pathways in largemouth bass virus-induced apoptosis and viral replication

作者:  Huang XH#, Wang W, Huang YH, Xu LW, Qin QW*.

刊物：Fish & Shellfish Immunology, (IF=3.034, I区)

刊号:  2014,41:371-379

影响因子:  3.034

摘要: Increased reports demonstrated that largemouth Bass, Micropterus salmoides in natural and artificial environments were always suffered from an emerging iridovirus disease, largemouth Bass virus (LMBV). However, the underlying mechanism of LMBV pathogenesis remained largely unknown. Here, we investigated the cell signaling events involved in virus induced cell death and viral replication in vitro. We found that LMBV infection in epithelioma papulosum cyprini (EPC) cells induced typical apoptosis, evidenced by the appearance of apoptotic bodies, cytochrome c release, mitochondrial membrane permeabilization (MMP) destruction and reactive oxygen species (ROS) generation. Two initiators of apoptosis, caspase-8 and caspase-9, and the executioner of apoptosis, caspase-3, were all significantly activated with the infection time, suggested that not only mitochondrion-mediated, but also death receptor-mediated apoptosis were involved in LMBV infection. Reporter gene assay showed that the promoter activity of transcription factors including p53, NF-kB, AP-1 and cAMP response elementbinding protein (CREB) were decreased during LMBV infection. After treatment with different signaling pathway inhibitors, virus production were significantly suppressed by the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway and extracellular-signal-regulated kinases (ERK) signaling pathway. Furthermore, LMBV infection induced apoptosis was enhanced by PI3K inhibitor LY294002, but decreased by addition of ERK inhibitor UO126. Therefore, we speculated that apoptosis was sophisticatedly regulated by a series of cell signaling events for efficient virus propagation. Taken together, our results provided new insights into the molecular mechanism of ranavirus infection.

题目: Involvement of  fish signal transducer and activator of transcription 3 (STAT3) in SGIV replication and virus induced paraptosis

作者:  Huang XH, Huang YH, Yang Y, Wei SN, Qin QW*

刊物：Fish & Shellfish Immunology, (IF=3.034, I区)

刊号:  2014,41:308-316

摘要: Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor which plays crucial roles in immune regulation, inflammation, cell proliferation, transformation, and other physiological processes of the organism. In this study, a novel STAT3 gene from orange spotted grouper (Ec-STAT3) was cloned and characterized. Bioinformatic analysis revealed that full-length of Ec-STAT3 was 3105-bp long and contained a 280-bp 50UTR, a 470-bp 30UTR, and a 2355-bp open reading frame (ORF) that encoded a 784-amino acid peptide. The deduced protein of Ec-STAT3 showed 98% identity to that of turbot (Scophthalmus maximus). Amino acid alignment showed that Ec-STAT3 contained four conserved domains, including a protein interaction domain, a coiled coil domain, a DNA binding domain, and an SH2 domain. Quantitative real-time PCR analysis showed that the highest expression level was detected in the liver, followed by skin and spleen. After injection with Singapore grouper iridovirus (SGIV), the transcript of Ec-STAT3 in spleen was increased significantly. To further explore the function of Ec-STAT3, we investigated the roles of Ec-STAT3 in SGIV infection in vitro. Immune fluorescence analysis indicated that SGIV infection altered the distribution of phosphorylated Ec-STAT3 in nucleus, and a small part of phosphorylated Ec-STAT3 was associated with virus assembly sites, suggesting that Ec-STAT3 might be important for SGIV infection. Using STAT3 specific inhibitor, S3I-201, we found that inhibition of Ec-STAT3 activation decreased the SGIV replication significantly. Moreover, inhibition of Ec-STAT3 activation obviously altered SGIV infection induced cell cycle arrest and the expression of pro-survival genes, including Bcl-2, Bcl-xL and Bax inhibitor. Together, our results firstly demonstrated the critical roles of fish STAT3 in DNA virus replication and virus induced paraptosis, but also provided new insights into the mechanism of iridovirus pathogenesis.