Comparative transient expression assay analysis of hycu-hr6- and IE1-dependent regulation of baculovirus gp64 early promoters in three insect cell lines
Comparative transient expression assay analysis of hycu-hr6- and IE1-dependent regulation of baculovirus gp64 early promoters in three insect cell lines
复制标题
DOI：
10.1016/j.virusres.2010.08.025
复制
发表时间：
2011-01-01
期刊：
影响因子：
5
通讯作者：
Ikeda, Motoko
中科院分区：
文献类型：
--
作者：
Nagai, Setsuko;Alves, Cristiano A. Felipe;Ikeda, Motoko
我们之前已经证明，美国棘球蚴多衣壳核多角体病毒（HycuMNPV） gp64基因（hycu-gp64）在病毒基因组上有一个1582 bp的大同源区域hycu-hr6，紧邻hycu-gp64基因的上游。本研究比较了顺式hycu-hr6和反式ie1在三种细胞系（Spodoptera frugiperda Sf9）中对HycuMNPV、Autographa cafornica multicapsid NPV （AcMNPV）和Bombyx mod NPV (BmNPV) gp64早期启动子的调控作用。Bombyx mod BM-N和Spilosoma imparilis SpIm)。含有报告荧光素酶基因的质粒的瞬时表达实验表明，gp64早期启动子受hycu-hr6的正调控，不受病毒和细胞类型的影响。相比之下，gp64早期启动子受ie1的正向或负向调控，以细胞和病毒型依赖的方式，表明细胞因子和病毒因子都负责ie1依赖性gp64早期启动子的调控。然而，hycu-gp64早期启动子活性一直被HycuMNPV IE1 （Hycu-IE1）抑制，与使用的细胞系无关。对hycu-gp64早期启动子区域的分析发现了两个新的序列元件，它们参与hycu- ie1依赖性的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弥补，但不能被AcMNPV - IE1结合基序（Ac-IBM）所取代。这些结果表明，IE1调节gp64早期启动子产生适量的gp64蛋白，这取决于npv -昆虫细胞系统。(C) 2010 Elsevier B.V.版权所有
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 1582 bp, hycu-hr6, immediately upstream of the hycu-gp64 gene. In the present study, we compared the regulation of gp64 early promoters from HycuMNPV, Autographa cahfornica multicapsid NPV (AcMNPV) and Bombyx mod NPV (BmNPV) by cis-acting hycu-hr6 and trans-acting IE1s in three cell lines (Spodoptera frugiperda Sf9. Bombyx mod 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. (C) 2010 Elsevier B.V. All rights reserved.