Herpes simplex virus protein VP16 activates immediate-early (IE) viral gene expression upon infection. VP16-mediated transactivation depends on formation of a multi protein complex with cellular factors on a cis-acting TAATGARA T sequence present in the IE promoters. The potent acidic activation domain, contained within the carboxyl terminus of VP16, is dispensable for the complex formation. The amino terminal part of VP16, which is inert in transactivation in mammalian cells, is sufficient for selective interactions with cellular factors, one of which has been identified as the ubiquitous transcription factor Oct-1. The yeast two-hybrid system was utilized to isolate the cellular factor(s) necessary in addition to Oct-1 for VP16 induced complex formation. This system, designed to directly clone proteins interacting with a given protein of interest, employs the yeast transcriptional activator GAL4. An interaction between VP16 and the cellular factor(s), fused to GAL4 DNA binding and activation domain, respectively, reconstitutes a hybrid transactivator that stimulates expression of a reporter lacZ gene in yeast. Thus, (beta)-galactosidase activity serves as a positive signal for protein-protein interaction. As a prelude of using this method for isolation of VP16-interacting cellular proteins, the system was tested with HSV-1 protein vhs, known to bind to VP16 in vitro. The obtained data demonstrated an interaction between VP16 and vhs in the two-hybrid system and deletion analysis revealed that VP16 sequence contained within the first 369 amino acids is required for binding to vhs. Thus, VP16 residues necessary for interaction with vhs in vivo coincide with these identified previously for VP16-vhs complex formation in vitro. VP16 fused to the GAL4 DNA binding domain activated expression of the reporter lacZ gene in yeast, despite the absence of its acidic activation domain. Deletion analysis showed that the amino terminal 369 residues of VP16 were sufficient for transactivation in yeast. Similar GAL4-VP16 derivatives were inactive in mammalian cells as measured by transient transfection assays. Thus, unlike in yeast, VP16 lacking the acidic activation domain is deficient in transactivation in mammalian cells even if it is directly bound to a promoter. VP16 sequences required for complex formation with vhs overlaps with those implicated in interaction with the mammalian factors, indicating that this region is involved in protein-protein interactions with both cellular and viral factors. Consistent with this, VP16 interaction with a yeast factor supplying an activation domain in trans would explain VP16-dependent transactivation in the absence of its acidic activation domain. Alternatively, a yeast specific activation domain might be present in the amino terminal part of VP16. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23354 |
| Date | 11 1900 |
| Creators | Popova, Bilyana |
| Contributors | Capone, John, Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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