A regulatory element involved in the transcriptional activation of the major capsid protein (VP5) of herpes simplex virus type 1 was identified and characterized in this research project. Gel mobility shift assay with nuclear extracts from both uninfected and HSV-1 infected HeLa cells identified two major protein-DNA complexes involving the VP5 promoter. No viral specific complex found. DNase I and orthophenanthroline-cu+ footprint analyses in the same laboratory revealed that the two complexes involve a single binding site, GGCCATCTTGAA, located between -64 and -75 bp relative to the VP5 cap site. To determine the function of this leaky-late binding site (LBS) in VP5 gene activation, mutated VP5 promoters with deletion and insertion around LBS were constructed and linked to a reporter gene, bacterial chloramphenicol acetyltransferase gene. The effect of mutations were tested in transient expression assay. Deletion of LBS resulted in seven to eight-fold reduction in the level of transactivation of the chloramphenicol acetyltransferase gene by superinfection with HSV-1 or by cotransfection of HSV immediate-early genes. These results indicated LBS is involved in the maximal transactivation of the VPS gene. A search of published gene sequences found the homologs of LBS exist in a number of HSV-1 By promoters, and other viral promoters, as well as cellar promoters. Some of these homologs have found involved in the transcription regulation.
| Identifer | oai:union.ndltd.org:pdx.edu/oai:pdxscholar.library.pdx.edu:open_access_etds-5787 |
| Date | 06 July 1994 |
| Creators | Chen, Shin |
| Publisher | PDXScholar |
| Source Sets | Portland State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations and Theses |
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