Baculovirus proteins IE-1, LEF-3, and P143 interact with DNA in vivo: a formaldehyde cross-linking study.
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.
Ito
Emma
E
Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Sahri
Daniela
D
Knippers
Rolf
R
Carstens
Eric B
EB
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002586:Cesium; D002712:Chlorides; D003593:Cytoplasm / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D004279:DNA, Viral / Q000096:biosynthesis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005557:Formaldehyde; D017874:Immediate-Early Proteins / Q000378:metabolism*; D047468:Immunoprecipitation; D017924:Nucleopolyhedroviruses / Q000378:metabolism*; D018411:Spodoptera; D015534:Trans-Activators / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
C069183:Ac-IE-1 protein, Autographa californica nucleopolyhedrovirus; D002712:Chlorides; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D015534:Trans-Activators; D014764:Viral Proteins; C094647:late expression factor 3, baculovirus; D005557:Formaldehyde; D002586:Cesium; D004265:DNA Helicases; C028019:cesium chloride