Liu Wang-Jing, Lo Chu-Fang, Kou Guang-Hsiung, Leu Jiann-Horng, Lai Ying-Jang, Chang Li-Kwan, Chang Yun-Shiang
Department of Earth and Life Science, College of Science, University of Taipei, Taipei 100, Taiwan.
Institute of Bioinformatics and Biosignal Transduction, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan.
Dev Comp Immunol. 2015 Mar;49(1):7-18. doi: 10.1016/j.dci.2014.10.015. Epub 2014 Nov 6.
A series of deletion and mutation assays of the white spot syndrome virus (WSSV) immediate-early gene WSSV108 promoter showed that a Krüppel-like factor (KLF) binding site located from -504 to -495 (relative to the transcription start site) is important for the overall level of WSSV108 promoter activity. Electrophoretic mobility shift assays further showed that overexpressed recombinant Penaeus monodon KLF (rPmKLF) formed a specific protein-DNA complex with the (32)P-labeled KLF binding site of the WSSV108 promoter, and that higher levels of Litopenaeus vannamei KLF (LvKLF) were expressed in WSSV-infected shrimp. A transactivation assay indicated that the WSSV108 promoter was strongly activated by rPmKLF in a dose-dependent manner. Lastly, we found that specific silencing of LvKLF expression in vivo by dsRNA injection dramatically reduced both WSSV108 expression and WSSV replication. We conclude that shrimp KLF is important for WSSV genome replication and gene expression, and that it binds to the WSSV108 promoter to enhance the expression of this immediate-early gene.
对白斑综合征病毒（WSSV）即刻早期基因WSSV108启动子进行的一系列缺失和突变分析表明，位于-504至-495（相对于转录起始位点）的一个类Krüppel因子（KLF）结合位点对于WSSV108启动子活性的整体水平很重要。电泳迁移率变动分析进一步表明，过表达的重组凡纳滨对虾KLF（rPmKLF）与WSSV108启动子的（32）P标记的KLF结合位点形成了特异性蛋白质-DNA复合物，并且在感染WSSV的虾中表达了更高水平的南美白对虾KLF（LvKLF）。一项反式激活分析表明，rPmKLF以剂量依赖性方式强烈激活WSSV108启动子。最后，我们发现通过注射dsRNA在体内特异性沉默LvKLF表达可显著降低WSSV108表达和WSSV复制。我们得出结论，对虾KLF对WSSV基因组复制和基因表达很重要，并且它与WSSV108启动子结合以增强这个即刻早期基因的表达。
