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Autographa californica nuclear polyhedrosis virus p143 gene product is a DNA-binding protein
1Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada.
Abstract:
We have identified the protein product of the Autographa californica nuclear polyhedrosis virus (AcMNPV) p143 gene by constructing a recombinant baculovirus overexpressing the gene product P143. The overexpressed protein exhibited a relative mobility of approximately 140 kDa and was stable for at least 12 hr after synthesis. Immunoblotting using a monoclonal antibody developed against the overexpressed protein identified a similar polypeptide in AcMNPV-infected cells which was detectable by 4 hr postinfection. P143 was present within infected cell nuclei at relatively constant amounts until at least 72 hr after infection, suggesting that P143 may perform other functions at late times after infection. P143, purified from infected cell nuclei by chromatography over hydroxylapatite and DNA cellulose, bound in a sequence-independent fashion to double-stranded but not to single-stranded DNA to form a ladder of retarded protein-DNA complexes. Together, these data are consistent with the essential role of P143 for viral DNA replication and suggest that P143 may function by direct binding to DNA.
Insights
Researchers identified the Autographa californica nuclear polyhedrosis virus (AcMNPV) P143 protein, essential for viral DNA replication. This protein binds directly to double-stranded DNA, suggesting a key role in viral processes.
Area of Science:
- Molecular Virology
- Insect Pathology
Background:
- Autographa californica nuclear polyhedrosis virus (AcMNPV) is a significant pathogen affecting insects.
- Understanding viral gene products is crucial for elucidating viral replication mechanisms.
Purpose of the Study:
- To identify and characterize the protein product of the AcMNPV p143 gene.
- To investigate the function and DNA-binding properties of the P143 protein.
Main Methods:
- Construction of a recombinant baculovirus for P143 overexpression.
- Protein characterization using SDS-PAGE and immunoblotting with monoclonal antibodies.
- Purification of P143 via hydroxylapatite and DNA cellulose chromatography.
- Analysis of P143 DNA-binding using electrophoretic mobility shift assays.
Main Results:
- The AcMNPV p143 gene product (P143) was overexpressed as a ~140 kDa protein, stable for over 12 hours.
- P143 was detected in AcMNPV-infected cells starting at 4 hours post-infection and persisted in the nucleus.
- Purified P143 bound double-stranded DNA in a sequence-independent manner, forming protein-DNA complexes.
Conclusions:
- The P143 protein is essential for AcMNPV DNA replication.
- P143 likely functions by directly binding to viral DNA during replication.