IE-1, LEF-3, and P143 are three of six proteins encoded by Autographa californica nucleopolyhedrovirus (AcMNPV) essential for baculovirus DNA replication in transient replication assays. IE-1 is the major baculovirus immediate early transcription regulator. LEF-3 is a single-stranded DNA binding protein (SSB) and P143 is a DNA helicase protein. To investigate their interactions in vivo, we treated AcMNPV-infected Spodoptera frugiperda cells with formaldehyde and separated soluble proteins from chromatin by cell fractionation and cesium chloride equilibrium centrifugation. Up to 70% of the total LEF-3 appeared in the fraction of soluble, probably nucleoplasmic proteins, while almost all P143 and IE-1 were associated with viral chromatin in the nucleus. This suggests that LEF-3 is produced in quantities that are higher than needed for the coverage of single stranded regions that arise during viral DNA replication and is consistent with the hypothesis that LEF-3 has other functions such as the localization of P143 to the nucleus. Using a chromatin immunoprecipitation procedure, we present the first direct evidence of LEF-3, P143, and IE-1 proteins binding to closely linked sites on viral chromatin in vivo, suggesting that they may form replication complexes on viral DNA in infected cells.

The baculovirus protein P143 is essential for viral DNA replication in vivo, likely as a DNA helicase. We have demonstrated that another viral protein, LEF-3, first described as a single-stranded DNA binding protein, is required for transporting P143 into the nuclei of insect cells. Both of these proteins, along with several other early viral proteins, are also essential for DNA replication in transient assays. We now describe the identification, nucleotide sequences, and transcription patterns of the Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) homologues of p143 and lef-3 and demonstrate that CfMNPV LEF-3 is also responsible for P143 localization to the nucleus. We predicted that the interaction between P143 and LEF-3 might be critical for cross-species complementation of DNA replication. Support for this hypothesis was generated by substitution of heterologous P143 and LEF-3 between two different baculovirus species, Autographa californica nucleopolyhedrovirus and CfMNPV, in transient DNA replication assays. The results suggest that the P143-LEF-3 complex is an important baculovirus replication factor.The family Baculoviridae represents a unique group of large rod-shaped enveloped viruses carrying a double-stranded circular DNA genome and replicating only in invertebrates. Many of the advances in understanding the molecular biology of baculoviruses have resulted from studies of variants of the type species Autographa californica nucleopolyhedrovirus (AcMNPV). Nucleopolyhedroviruses (NPVs) replicate in cell nuclei and are characterized by the production of two virion phenotypes, the budded virions and the occlusion-derived virions. Both forms are produced following infection of cells in culture and are characteristic of late stages of the viral replication cycle following initiation of viral DNA replication at about 8 h postinfection (37). The early events prior to this time are characterized by the expression of several viral gene products, some of which have been shown to be essential for viral DNA replication. Nine viral genes (ie-1, ie-2, p143, dnapol, lef-1, lef-2, lef-3, pe38, and p35) are involved in directing replication of plasmids carrying viral DNA inserts in transfected cells (20,31,38). These data supported earlier genetic analysis of a conditional lethal AcMNPV mutant defective in DNA replication (13), which led to the description of the p143 gene: its nucleotide sequence and the identification of the lesion in the 1,221-amino-acid open reading frame (ORF) (143 kDa) responsible for the temperature-sensitive DNA negative phenotype (29). The p143 gene is essential for viral DNA replication in vivo since no replication occurs in cells infected at the nonpermissive temperature with ts8 (29).Biochemical characterization of extracts from AcMNPV-infected cells showed that P143 copurified through hydroxylapatite and coeluted from single-stranded DNA cellulose with another viral protein called LEF-3, suggesting a possible direct interaction between P143 and LEF-3 (22, 39). LEF-3, also demonstra...