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. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-05-27 15:02:53.983
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/6531 |
| Date | 27 May 2011 |
| Creators | Adetola, Gbolagade |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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