Nuclear IE2 Structures Are Related to Viral DNA Replication Sites during Baculovirus Infection

Abstract

ABSTRACT The ie2 gene of Autographa californica multicapsid nuclear polyhedrosis virus is 1 of the 10 baculovirus genes that have been identified as factors involved in viral DNA replication. IE2 is detectable in the nucleus as one of the major early-expressed proteins and exhibits a dynamic localization pattern during the infection cycle (D. Murges, I. Quadt, J. Schröer, and D. Knebel-Mörsdorf, Exp. Cell Res. 264:219-232, 2001). Here, we investigated whether IE2 localized to regions of viral DNA replication. After viral DNA was labeled with bromodeoxyuridine (BrdU), confocal imaging indicated that defined IE2 domains colocalized with viral DNA replication centers as soon as viral DNA replication was detectable. In addition, a subpopulation of IE2 structures colocalized with two further virus-encoded replication factors, late expression factor 3 (LEF-3) and the DNA binding protein (DBP). While DBP and LEF-3 structures always colocalized and enlarged simultaneously with viral DNA replication sites, only those IE2 structures that colocalized with replication sites also colocalized with DBP. Replication and transcription of DNA viruses in association with promyelocytic leukemia protein (PML) oncogenic domains have been observed. By confocal imaging we demonstrated that the human PML colocalized with IE2. Triple staining revealed PML/IE2 domains in the vicinity of viral DNA replication centers, while IE2 alone colocalized with early replication sites, demonstrating that PML structures do not form common domains with viral DNA replication centers. Thus, we conclude that IE2 colocalizes alternately with PML and the sites of viral DNA replication. Small ubiquitin-like modifier SUMO-1 has been implicated in the nuclear distribution of PML. Similar to what was found for mammalian cells, small ubiquitin-like modifiers were recruited to PML domains in infected insect cells, which suggests that IE2 and PML colocalize in conserved cellular domains. In summary, our results support a model for IE2 as part of various functional sites in the nucleus that are connected with viral DNA replication.