Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
Virus Res. 2011 Jan;155(1):83-90. doi: 10.1016/j.virusres.2010.08.025. Epub 2010 Sep 17.
We previously demonstrated that the Hyphantria cunea multicapsid nucleopolyhedrovirus (HycuMNPV) gp64 gene (hycu-gp64) is uniquely localized on the viral genome with a large homologous region of 1582bp, hycu-hr6, immediately upstream of the hycu-gp64 gene. In the present study, we compared the regulation of gp64 early promoters from HycuMNPV, Autographa californica multicapsid NPV (AcMNPV) and Bombyx mori NPV (BmNPV) by cis-acting hycu-hr6 and trans-acting IE1s in three cell lines (Spodoptera frugiperda Sf9, Bombyx mori BM-N and Spilosoma imparilis SpIm). A transient expression assay with plasmids harboring a reporter luciferase gene demonstrated that the gp64 early promoters are positively regulated by hycu-hr6, independent of virus and cell types. In contrast, gp64 early promoters were regulated positively or negatively by trans-acting IE1s, in a cell- and virus-type dependent manner, indicating that cellular factors, as well as viral factors, are responsible for IE1-dependent regulation of gp64 early promoters. However, hycu-gp64 early promoter activity was consistently suppressed by HycuMNPV IE1 (Hycu-IE1), irrespective of the cell lines used. Analysis of the hycu-gp64 early promoter region revealed two novel sequence elements that were involved in Hycu-IE1-dependent negative regulation of the hycu-gp64 early promoter. These two novel regulatory sequence elements could compensate for each other but could not be substituted with AcMNPV IE1 binding motif (Ac-IBM). These results suggest that IE1 regulates gp64 early promoters to produce the proper amount of GP64 protein, depending upon NPV-insect cell systems.
我们之前的研究表明，舞毒蛾多角体核型多角体病毒（HycuMNPV）的 gp64 基因（hycu-gp64）在病毒基因组上具有独特的定位，其上游紧邻一个含有 1582bp 的大片段同源区域 hycu-hr6。在本研究中，我们比较了 HycuMNPV、苜蓿银纹夜蛾多角体病毒（AcMNPV）和家蚕核型多角体病毒（BmNPV）的 gp64 早期启动子在三种细胞系（草地贪夜蛾 Sf9、家蚕 BM-N 和斜纹夜蛾 SpIm）中的调控作用，这些启动子由顺式作用元件 hycu-hr6 和反式作用元件 IE1 调控。含有报告基因荧光素酶的瞬时表达实验表明，gp64 早期启动子受 hycu-hr6 的正向调控，与病毒和细胞类型无关。相比之下，gp64 早期启动子受转座激活因子 IE1 的正向或负向调控，这种调控依赖于细胞类型和病毒类型，这表明细胞因子和病毒因子都负责 IE1 对 gp64 早期启动子的调控。然而，HycuMNPV IE1（Hycu-IE1）始终抑制 HycuMNPV 的 gp64 早期启动子活性，而与所使用的细胞系无关。对 hycu-gp64 早期启动子区域的分析揭示了两个新的序列元件，它们参与了 Hycu-IE1 对 hycu-gp64 早期启动子的负调控。这两个新的调控序列元件可以相互补偿，但不能被 AcMNPV IE1 结合基序（Ac-IBM）取代。这些结果表明，IE1 根据杆状病毒-昆虫细胞系统来调节 gp64 早期启动子，以产生适量的 GP64 蛋白。
