Focal Distribution of Baculovirus IE1 Triggered by Its Binding to the hr DNA Elements

Abstract

In BmN cells infected with the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV), IE1, a principal transcriptional activator, localizes to sites of viral DNA replication. IE1 initially displays focal distribution in BmNPV-infected cells prior to DNA synthesis, whereas the protein expressed by transfection with the ie1 gene is distributed throughout the nucleoplasm instead of localized to discrete subnuclear structures. To identify the inducer of focus formation for IE1, we conducted transfection experiments with an IE1-GFP construct and found that cotransfection with genomic DNA fragments bearing the homologous region (hr) sequences caused the formation of IE1-green fluorescent protein (GFP) foci. The transfection of insect cells with a single plasmid containing exclusively the hr3 sequence and the IE1-GFP gene was sufficient to form IE1-GFP foci. These results suggest that hr elements are a primary determinant of the focal distribution of IE1. An analysis of a series of hr3 deletion mutants showed that a single copy of the direct repeat could induce the formation of IE1 foci. Targeted mutagenesis within the hr-binding domain of IE1-GFP caused impairment of the hr-dependent IE1 localization, suggesting that binding of IE1 to the hr elements is essential for the onset of IE1 focus formation. The observation of BmNPV IE1 foci in non-BmNPV-susceptible cells suggests that no speciesspecific factors are required for hr-dependent IE1 focus formation.The immediate early gene 1 (ie1) of Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a 67-kDa nuclear protein (IE1) that plays crucial roles in viral replication (reviewed in reference 6). In BmNPV-infected BmN cells, IE1 localizes to discrete subnuclear structures in which viral DNA synthesis occurs (26). While IE1 was originally characterized as a transcriptional activator of baculovirus early gene expression (2,3,10,11,13,17,18,19,25,30,31,34), it has also been demonstrated to be essential for the replication of plasmids carrying putative origins of virus DNA replication, known as homologous region (hr) sequences (1,15,21).hr sequence elements have been identified in the genomes of many baculoviruses (see reference 20). The BmNPV genome possesses five hr elements, which are dispersed at map units (m.u.) ϳ18 (hr2-left), ϳ19 (hr2-right), ϳ51 (hr3), ϳ67.5 (hr4-left), ϳ70 (hr4-right), ϳ83.5 (hr5), and ϳ96.5 (hr1) on the ϳ128-kbp genome (23). The BmNPV hrs are characterized by a series of ϳ75-bp DNA repeats, with each containing a 30-bp imperfect palindrome with an EcoRI site at its core (23). When viral early genes are cis-linked to the hr sequences, IE1-mediated activation of their transcription is greatly enhanced (9, 12, 25). Gel shift assays and mutational analyses have shown that IE1 binds directly to the hr sequences, which serve both as enhancers and as putative replication origins (3,4,8,12,16,25,28,32,33). It has also been reported that the palindrome in the hr sequences is essential for the direct interaction between the hr sequences and the IE1 protein (5,7,18...

Transactivator IE1 Is Required for Baculovirus Early Replication Events That Trigger Apoptosis in Permissive and Nonpermissive Cells

“…Transactivation by IE1 is enhanced by its binding as a homodimer to the baculovirus homologous region (hr) sequences, which function as transcriptional enhancers and origins of viral DNA replication (5,22,28,42,45,50). The capacities of IE1 to interact with origins of viral DNA replication and to associate with DNA replication factories are consistent with the postulated role of IE1 in baculovirus DNA replication (5,19,28,36,50). Thus, IE1 appears to have multiple in vivo functions, as originally suggested by the variety of behaviors displayed by a temperature-sensitive mutant of IE1 (49).…”

Nuclear Marginalization of Host Cell Chromatin Associated with Expansion of Two Discrete Virus-Induced Subnuclear Compartments during Baculovirus Infection

“…This result indicates a correlation between formation of the IE1/LEF3/P143-associated structures and chromatin exclusion. Omission of LEF3 or P143 induced formation of IE1 foci instead of the IE1/LEF3/P143-associated structures because only IE1 and hr, and not all four genes, were present in the transfected cells (15,16). In the transfected cells chromatin was not excluded from the IE1 foci (Fig.…”

Dissection of two modes of IE1 sub-nuclear localization in baculovirus-infected cells

“…Confocal microscopy revealed a focal distribution pattern for IE1 within the nucleus prior to viral DNA replication that then apparently expands and develops into the VS during DNA replication (Kawasaki et al, 2004;Okano et al, 1999). This observation suggests that IE1 foci may function as the primary scaffold for VS (Nagamine et al, 2005(Nagamine et al, , 2006.…”