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http://hdl.handle.net/11375/8671
Title: | Investigation of the mechanisms of transcription and transactivation by the herpes simplex virus protein Vmw65 |
Authors: | Werstuck, Hamilton Geoffrey |
Advisor: | Capone, John P. |
Department: | Biochemistry |
Keywords: | Biochemistry;Biochemistry |
Publication Date: | 1993 |
Abstract: | <p>Eukaryotic gene expression is regulated through the assembly of transcription factors into multi-component complexes that interact with cis-acting DNA recognition sites. The herpes simplex virus protein, Vmw65 (also known as VP16, αTIF), stimulates transcription through direct and indirect contacts with specific cellular factors (including the ubiquitous octamer binding protein, Oct-1) and a conserved enhancer-like DNA sequence (consensus TAATGARAT) found upstream of the viral immediate-early (IE) genes. The objective of this project was to utilize this viral system as a model to investigate mechanisms by which eukaryotic gene transcription is regulated. The functional interactions of Vmw65 with the cellular transcription machinery make this a valuable system for the elucidation of the protein-protein and protein-DNA interactions involved in regulation. This thesis describes various strategies that have been utilized to examine the different aspects Vmw65 mediated transactivation. The structure-function profile of Vmw65 was determined through the construction of a series of mutants that were individually assayed for specific activities. An in vivo transient transfection system was utilized to measure the ability of each of the mutants to transactivate an indicator gene, containing a TAATGARAT sequence, relative to the wild type protein. This assay was also utilized to map domains of Vmw65 that are capable of interfering with the wild type transactivation function in a trans-dominant manner. Finally, the mutants were expressed in E. coli as protein A fusion proteins and each was analyzed for its ability to direct the assembly of the Vmw65 dependent complex and to bind directly to DNA in the absence of other factors. To summarize, the transactivation function is highly sensitive to mutations within two distinct internal regions of the protein (corresponding to amino acids 178-215 and 335-379). A region of Vmw65, located between amino acids 141 and 186, is capable of interfering with the wild type transactivation function in vivo and is required for complex assembly in vitro. These results suggest that this domain forms essential contacts with a factor involved in the transactivation function of Vmw65. The transcription activation domain of Vmw65 is not required for complex assembly indicating that Vmw65 is a modular protein consisting of a transcription activation domain and a complex assembly domain. Each of these domains can independently impart its activity to a heterologous protein. Additional cellular components of the Vmw65 dependent complex were identified and characterized using cell fractionation techniques. One factor, designated SF, was shown to specifically stabilize the multi-protein complex. SF has an apparent molecular weight of 1500-3000 Da and is resistant to heat treatment as well as extensive digestions with protease, nuclease and phospholipase. β-glucuronidase digestion did have an effect on SF activity suggesting that this factor may be composed, at least partially, of carbohydrate. Finally, the role of Vmw65 in the herpes simplex virus lytic cycle was investigated through the construction and characterization of a stable cell line (designated BSV65) expressing this transactivator. The cell line was shown to specifically activate viral immediate early genes and was capable of complementing a virus defective for the Vmw65 transactivation function. The transfection of purified HSV-1 DNA into BSV65 cells resulted in a 200 fold increase in virus production relative to the parental cell line. (Abstract shortened by UMI.)</p> |
URI: | http://hdl.handle.net/11375/8671 |
Identifier: | opendissertations/3857 4874 1737877 |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
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fulltext.pdf | 5.55 MB | Adobe PDF | View/Open |
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