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http://hdl.handle.net/11375/7101
Title: | Investigation of Protein-Protein and Protein-DNA Interactions Involved in the Function of Herpes Simplex Virus Transactivator Vmw65 |
Authors: | Shaw, Xiao Peter |
Advisor: | Capone, John P. |
Department: | Biochemistry |
Keywords: | Biochemistry;Biochemistry |
Publication Date: | Dec-1995 |
Abstract: | <p>The herpes simplex virus phosphoprotein Vmw65 (also referred to as VP16, αTIF), strongly stimulates transcription through recognition of the cis-acting consensus sequence, TAATGARAT (R=purine), which is present in upstream regions of viral immediate early genes in at least one copy. However, Vmw65 has only weak intrinsic DNA binding activity. In order to execute its function as an activator, Vmw65 mediates the assembly of a multi-component complex with host cell factors that include Oct-1. The objective of this project was to utilize HSV-1 as a model system to investigate mechanisms involved in transcriptional regulation of eukaryotic genes, in particular, to elucidate how viral and cellular proteins are selected and assembled into transcriptional active complexes at the cis-acting sites through protein-protein and protein-DNA interactions.</p> <p>Using affinity chromatography columns coupled with E. coli expressed protein A fused Vmw65, a novel cellular factor (referred to as VCAF-1), distinct from Oct-1, that bound directly to Vmw65 in the absence of target DNA was identified, and was found to be necessary for Vmw65-mediated complex assembly with Oct-1. This study also revealed that Vrnw65 does not form a complex, directly or indirectly, with Oct-1 in the absence of target DNA.</p> <p>Some distinct properties of Vmw65 involved in generating functionally active multi-component complexes and their requirements for complex assembly and activation were investigated in vitro using mobility shift assays with ³²p-labelled oligonucleotides and purified components, as well as in vivo using CAT assays with mutant derivatives.</p> <p>We found that Vmw65 has intrinsic DNA binding activity when used at a high concentration. This activity is sequence-specific and requires both regions 141-250 and 335-390 within the N-terminal complex forming domain (1-411). Vmw65 is able to cooperatively interact with DNA-bound Oct-1 in the absence of VCAF-1. The region containing amino acids 379-404 is required for cooperativity with Oct-1. The intrinsic DNA binding or cooperative interaction with DNA-bound Oct-1 may facilitate the assembly of multi-component complex. However, mutational analysis demonstrated that the abilities of Vmw65 to directly bind to DNA or interact with DNA-bound Oct-1 can be uncoupled from complex assembly and activation.</p> <p>The independent interaction between Vmw65 and VCAF-1 may playa regulatory role in complex assembly and activation. Our study found that the sequence requirements for Vmw65 to independently interact with VCAF-1 overlaps but is distinct from that for complex formation. Furthermore, using amino acid substitutions, we demonstrated that the direct interaction of Vmw65 with VCAF-1 is not necessarily required for the formation of the multi-component complex in vitro, nor for the activation of IE genes in vivo. Our findings suggest that more than one pathway exists in cooperative assembly of the Vmw65 induced complex, and that selective protein-protein interactions may play important roles in differentiating these pathways. This may allow HSV to adapt to various physiological conditions of infected cells and regulate its productive life cycle.</p> <p>The studies presented in this thesis have been reported, in part, in the following publications:</p> <p>1. Xiao, P. and Capone, J.P. (1990) A cellular factor binds to the herpes simplex virus type 1 transactivator Vmw65 and is required for Vmw6S-dependent protein-DNA complex assembly with Oct-1. Mol. Cel. BioI. 10, 4974-4977.</p> <p>2. Smibert, C.A., Popova, B., Xiao, P., Capone, J.P. and Smiley, J.R. (1994) Herpes simplex virus VP-16 forms a complex with the virion host shutoff protein. J. Virol. 68, 2339-2346.</p> <p>3. Popova, B., Bilan, P., Xiao, P., Faught, M. and Capone, J.P. (1994) Transcriptional activation by DNA-binding derivatives of HSV-1 VP16 that lack the carboxyl-terminal acidic activation domain. Virol. 209, 19-28.</p> <p>4. Shaw, P., Knez, J. and Capone, J.P. (1995) Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interactions and DNA binding from complex assembly and activation. J. Biol. Chem. 270, 29030-29037.</p> |
URI: | http://hdl.handle.net/11375/7101 |
Identifier: | opendissertations/2396 3334 1374751 |
Appears in Collections: | Open Access Dissertations and Theses |
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fulltext.pdf | 5.48 MB | Adobe PDF | View/Open |
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