Comparative transient expression assay analysis of hycu-hr6- and IE1-dependent regulation of baculovirus gp64 early promoters in three insect cell lines.

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.