CHARACTERIZATION OF THE BACULOVIRUS LATE EXPRESSION FACTOR-3 OLIGOMERIZATION INTERACTION DOMAINS USING PROTEIN COMPLEMENTATION ASSAY
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Late expression factor 3 is one of the six AcMNPV genes essential for DNA replication identified through transient replication assays. LEF-3 is a single stranded DNA binding protein responsible for the transportation of the viral helicase (P143) into the nucleus of the infected cell. In this study, a protein complementation-based assay was adapted to identify the region(s) of LEF-3 that is (are) involved in LEF-3-LEF-3 protein interactions. The full-length LEF-3, or various truncated LEF-3 regions were fused with Venus1 (N- terminus portions of full length Venus, a modified yellow fluorescence protein) or Venus2 (C- terminus). Venus1 and Venus2 fragments generated a functional fluorescent Venus protein when the two fragments were brought together by protein-protein interaction of the fused LEF-3 constructs. Fluorescence generated by coexpression of full-length LEF-3 fusion proteins confirmed that LEF-3 exists as homo-oligomer. Interaction between the full-length and the N- terminal (aa 1-189) or C- terminal regions (aa 190-385), and between the various truncated LEF-3 regions suggested the complexity of LEF-3 oligomeric structure. LEF-3 constructs deleted for NLS function revealed cytoplasmic fluorescence, suggesting that LEF-3-LEF-3 interactions occur in the absence of DNA or nuclear proteins. Because LEF-3 is essential for nuclear transporting the viral helicase (P143), the ability of LEF-3 to interact with another viral protein was investigated. P47, a sub-unit of the viral RNA polymerase was chosen because it is cytoplasmic when expressed on its own. The interaction between LEF-3 and P47 produced complete nuclear localized fluorescent signals. Overall, the results suggest that there are multiple regions of LEF-3 that are capable of closely interacting, and that multiple domains are likely involved in the oligomerization of full-length LEF-3. The interaction of LEF-3 with P47 suggests that P47 may be another LEF-3 cargo protein.