Institute of Zoology, College of Life Science, National Taiwan University, Taipei, Taiwan, Republic of China.
Antioxid Redox Signal. 2012 Sep 15;17(6):914-26. doi: 10.1089/ars.2011.4264. Epub 2012 Apr 16.
In this study we identified viral gene targets of the important redox regulator thioredoxin (Trx), and explored in depth how Trx interacts with the immediate early gene #1 (IE1) of the white spot syndrome virus (WSSV).
In a pull-down assay, we found that recombinant Trx bound to IE1 under oxidizing conditions, and a coimmunoprecipitation assay showed that Trx bound to WSSV IE1 when the transfected cells were subjected to oxidative stress. A pull-down assay with Trx mutants showed that no IE1 binding occurred when cysteine 62 was replaced by serine. Electrophoretic mobility shift assay (EMSA) showed that the DNA binding activity of WSSV IE1 was downregulated under oxidative conditions, and that Penaeus monodon Trx (PmTrx) restored the DNA binding activity of the inactivated, oxidized WSSV IE1. Another EMSA experiment showed that IE1's Cys-X-X-Cys motif and cysteine residue 55 were necessary for DNA binding. Measurement of the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) in WSSV-infected shrimp showed that oxidative stress was significantly increased at 48 h postinfection. The biological significance of Trx was also demonstrated in a double-strand RNA Trx knockdown experiment where suppression of shrimp Trx led to significant decreases in mortality and viral copy numbers.
WSSV's pathogenicity is enhanced by the virus' use of host Trx to rescue the DNA binding activity of WSSV IE1 under oxidizing conditions.
本研究鉴定了重要的氧化还原调节剂硫氧还蛋白（Trx）的病毒基因靶标，并深入探讨了 Trx 如何与白斑综合征病毒（WSSV）的早期基因 #1（IE1）相互作用。
在下拉测定中，我们发现还原条件下重组 Trx 与 IE1 结合，共免疫沉淀测定显示转染细胞受到氧化应激时 Trx 与 WSSV IE1 结合。用 Trx 突变体进行的下拉测定表明，当半胱氨酸 62 被丝氨酸取代时，IE1 没有结合。电泳迁移率变动分析（EMSA）表明氧化条件下 WSSV IE1 的 DNA 结合活性下调，而斑节对虾 Trx（PmTrx）恢复了失活、氧化的 WSSV IE1 的 DNA 结合活性。另一个 EMSA 实验表明，IE1 的 Cys-X-X-Cys 基序和半胱氨酸残基 55 是 DNA 结合所必需的。在感染 WSSV 的虾中测量还原型谷胱甘肽与氧化型谷胱甘肽（GSH/GSSG）的比值表明，感染后 48 小时氧化应激显著增加。在双链 RNA Trx 敲低实验中也证明了 Trx 的生物学意义，其中虾 Trx 的抑制导致死亡率和病毒拷贝数显著降低。
WSSV 通过利用宿主 Trx 在氧化条件下拯救 WSSV IE1 的 DNA 结合活性，增强了其致病性。
