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A baculovirus single-stranded DNA binding protein, LEF-3, mediates the nuclear localization of the putative helicase
1Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada.
Abstract:
The intracellular localization of the baculovirus Autographa californica nuclear polyhedrosis virus p143 gene product (P143) was investigated by immunofluoresence staining of infected and transfected cells. As expected for a protein essential for viral DNA replication, under these conditions, P143 was localized to the nucleus. However, when a plasmid directing the synthesis of P143 from its own promoter was co-transfected with a plasmid expressing IE-1, P143 was found in the cytoplasm. Cotransfection of cells with a series of plasmids expressing viral genes sufficient for stimulation of plasmid replication resulted in nuclear localization of P143 suggesting that one of the gene products required for viral DNA replication may also assist nuclear localization of P143. Sequential deletion of various genes from this assay revealed that when the plasmid expressing LEF-3 was deleted from the mixture, P143 remained in the cytoplasm. Plasmids were constructed where the synthesis of LEF-3 and P143 were directed by the ie-1 promoter. When these plasmids were cotransfected, both LEF-3 and P143 colocalized to the nucleus suggesting that an important function of LEF-3 is intracellular trafficking of P143 and directing it to the nucleus.
Insights
The baculovirus Autographa californica nuclear polyhedrosis virus p143 gene product (P143) is essential for viral DNA replication. LEF-3 protein facilitates P143
Area of Science:
- Molecular Virology
- Cell Biology
- Baculovirus Research
Background:
- Baculoviruses are important tools in molecular biology and biotechnology.
- Understanding viral protein localization is crucial for deciphering viral replication mechanisms.
- The p143 gene product (P143) of Autographa californica nuclear polyhedrosis virus (AcNPV) is vital for viral DNA replication.
Purpose of the Study:
- To investigate the intracellular localization of the AcNPV P143 protein.
- To identify viral factors that regulate P143 nuclear import.
- To elucidate the role of LEF-3 in P143 trafficking.
Main Methods:
- Immunofluorescence staining of infected and transfected insect cells.
- Co-transfection assays with plasmids expressing viral genes.
- Site-directed mutagenesis and gene deletion studies.
Main Results:
- P143 is primarily localized to the nucleus in infected cells, consistent with its role in DNA replication.
- Co-expression of AcNPV IE-1 with P143 resulted in cytoplasmic localization of P143.
- Nuclear localization of P143 was restored upon co-expression of viral replication genes, with LEF-3 identified as critical for this process.
- LEF-3 directly mediates the nuclear import of P143.
Conclusions:
- The intracellular localization of AcNPV P143 is regulated by viral factors.
- LEF-3 plays a crucial role in the nuclear trafficking and localization of P143.
- This interaction highlights a key mechanism in baculovirus DNA replication.