Analysis of baculovirus IE1 in living cells: dynamics and spatial relationships to viral structural proteins.

IE1, a principal transcriptional activator of the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV), is an essential factor for viral DNA replication. During viral infection, IE1 accumulates in discrete subnuclear structures where viral DNA replication occurs. To analyse the dynamic properties of IE1, we monitored green fluorescent protein-tagged IE1 (IE1-GFP) in BmNPV-infected B. mori cells by live-cell microscopy. Time-lapse imaging showed that IE1-associated structures gradually expanded and occasionally fused with one another, while photobleaching experiments revealed that IE1-GFP was relatively immobile inside the IE1-associated structures. To investigate the spatial relationships between IE1 and viral structural proteins in infected cells, three GFP-tagged viral components were expressed together with DsRed-tagged IE1. Two structural proteins that constitute the occlusion-derived virus (ODV), P91-GFP and GFP-ODV-E25, localized to the periphery of the IE1-associated structures. While local accumulations of these proteins were often in contact with the IE1-associated structures, they did not extend beyond the boundaries of the structures. In contrast, the major capsid protein VP39-GFP predominantly accumulated within the IE1-associated structures. These data indicated, in conjunction with the finding of a high DNA content in the structures, that IE1 localizes to the virogenic stroma and therefore support the prediction previously proposed that the virogenic stroma is a site for viral DNA replication as well as for the assembly of nucleocapsids.