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Reconstructing the replication complex of AcMNPV
Kathleen L Hefferon1, Lois K Miller
1Center for Virology, Department of Botany, University of Toronto, Ontario, Canada.
European Journal of Biochemistry
|December 11, 2002
Summary
Replication factors of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) were tagged and studied. Most factors localize to the nucleus, and their interactions suggest a viral replication complex.
Area of Science:
- Molecular Virology
- Insect Pathology
- Genomics
Background:
- Baculoviruses, like AcMNPV, possess large double-stranded DNA genomes.
- Viral DNA replication is crucial for activating late gene expression during infection.
Purpose of the Study:
- To investigate the expression, localization, and interactions of AcMNPV replication factors.
- To elucidate the formation of a potential AcMNPV replication complex.
Main Methods:
- FLAG- and HA-tagging of AcMNPV replication factors.
- CAT assays and Western blot analysis for expression and activity.
- Subcellular fractionation and immunofluorescent microscopy for localization.
- Yeast two-hybrid and co-precipitation for interaction studies.
Main Results:
- Most replication late expression factors (LEFs) were found in the nucleus, excluding P143 and P35.
- Interactions among replication LEFs were mapped using yeast two-hybrid and co-precipitation.
- A model for the AcMNPV replication complex was proposed based on interaction data.
Conclusions:
- Nuclear localization is a key feature for most AcMNPV replication LEFs.
- The identified interactions support a model of a coordinated viral replication complex.