BACKGROUND Iridoviruses are large DNA viruses that cause diseases in fish, amphibians and insects. Singapore grouper iridovirus (SGIV) is isolated from cultured grouper and characterized as a ranavirus. ICP46 is defined to be a core gene of the family Iridoviridae and SGIV ICP46 was demonstrated to be an immediate-early (IE) gene associated with cell growth control and could contribute to virus replication in previous research. METHODS The transcription start site (TSS) and 5'-untranslated region (5'-UTR) of SGIV ICP46 were determined using 5' RACE. The core promoter elements of ICP46s were analyzed by bioinformatics analysis. The core promoter region and the regulation model of SGIV ICP46 promoter were revealed by the construction of serially deleted promoter plasmids, transfections, drug treat and luciferase reporter assays. The identification of virion-associated transcriptional transactivator (VATT) that interact with SGIV ICP46 promoter and their binding site on promoter were performed by electrophoretic mobility shift assays (EMSA), DNA pull-down assays and mass spectrometry (MS). RESULTS SGIV ICP46 was found to have short 5'-UTR and a presumptive downstream promoter element (DPE), AGACA, which locates at + 36 to + 39 nt downstream of the TSS. The core promoter region of SGIV ICP46 located from - 22 to + 42 nt relative to the TSS. VATTs were involved in the promoter activation of SGIV ICP46 and further identified to be VP12, VP39, VP57 and MCP. A 10-base DNA sequence "ATGGCTTTCG" between the TSS and presumptive DPE was determined to be the binding site of the VATTs. CONCLUSION Our study showed that four VAATs (VP12, VP39, VP57 and MCP) might bind with the SGIV ICP46 promoter and be involved in the promoter activation. Further, the binding site of the VATTs on promoter was a 10-base DNA sequence between the TSS and presumptive DPE.

中文翻译：

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鉴定SGIV ICP46启动子的病毒体相关转录反式激活因子（VATT）及其在启动子上的结合位点。

背景技术虹膜病毒是引起鱼类，两栖动物和昆虫疾病的大DNA病毒。新加坡石斑鱼虹膜病毒（SGIV）是从培养的石斑鱼中分离出来的，并被表征为一种鼻病毒。ICP46被定义为Iridoviridae家族的核心基因，而SGIV ICP46被证明是与细胞生长控制相关的即早（IE）基因，并且在以前的研究中可能有助于病毒复制。方法使用5'RACE测定SGIV ICP46的转录起始位点（TSS）和5'-非翻译区（5'-UTR）。ICP46s的核心启动子元素通过生物信息学分析进行了分析。通过序列缺失的启动子质粒的构建，转染，药物处理和荧光素酶报告基因分析揭示了SGIV ICP46启动子的核心启动子区域和调控模型。通过电泳迁移率迁移分析（EMSA），DNA下拉分析和质谱（MS）鉴定了与SGIV ICP46启动子相互作用的病毒体相关转录反式激活因子（VATT）及其在启动子上的结合位点。结果发现SGIV ICP46具有短的5'-UTR和一个推测的下游启动子元件（DPE）AGACA，其位于TSS下游的+36至+39 nt。SGIV ICP46的核心启动子区域相对于TSS位于-22到+ 42 nt之间。VATTs参与了SGIV ICP46的启动子激活，并进一步确定为VP12，VP39，VP57和MCP。TSS和推定的DPE之间的10个碱基的DNA序列“ ATGGCTTTCG”被确定为VATT的结合位点。结论我们的研究表明，有四个VAAT（VP12，VP39，VP57和MCP）可能与SGIV ICP46启动子结合并参与启动子激活。此外，VATTs在启动子上的结合位点是TSS和推测的DPE之间的10个碱基的DNA序列。