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Baculovirus proteins IE-1, LEF-3, and P143 interact with DNA in vivo: a formaldehyde cross-linking study
Emma Ito1, Daniela Sahri, Rolf Knippers
1Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Abstract:
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.
Insights
Autographa californica nucleopolyhedrovirus (AcMNPV) proteins IE-1, LEF-3, and P143 interact in vivo. These essential baculovirus DNA replication proteins bind viral chromatin, suggesting they form replication complexes.
Area of Science:
- Molecular biology
- Virology
- Cell biology
Background:
- Autographa californica nucleopolyhedrovirus (AcMNPV) encodes six essential DNA replication proteins.
- IE-1 is a key transcriptional regulator, LEF-3 is a single-stranded DNA binding protein (SSB), and P143 is a DNA helicase.
Purpose of the Study:
- To investigate the in vivo interactions of AcMNPV proteins IE-1, LEF-3, and P143.
- To determine the cellular localization and DNA binding of these essential replication proteins.
Main Methods:
- Formaldehyde crosslinking of infected insect cells (Spodoptera frugiperda).
- Cell fractionation and cesium chloride equilibrium centrifugation to separate soluble and chromatin-bound proteins.
- Chromatin immunoprecipitation (ChIP) to detect in vivo protein-DNA interactions.
Main Results:
- LEF-3 is primarily soluble (nucleoplasmic), while P143 and IE-1 are predominantly chromatin-associated.
- LEF-3 is present in higher quantities than needed for DNA binding alone, suggesting additional roles.
- Direct evidence of IE-1, LEF-3, and P143 binding to viral chromatin in vivo was obtained.
Conclusions:
- IE-1, LEF-3, and P143 likely form functional replication complexes on viral DNA within infected cells.
- LEF-3 may play a role in localizing P143 to the nucleus, beyond its SSB function.
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