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The herpes simplex virus 1 (HSVl) VP5 gene codes for the major viral capsid protein. Understanding of the mechanism of how the VP5 gene is regulated in host cells will help us to understand the molecular action of the HSV 1 life cycle and its interplay with the host cell gene expression machinery (transcription and translation). This may ultimately provide scientific bases for both better prevention and cure of HSV 1 caused diseases. Previous work from Dr. Robert L. Millette' s laboratory has indicated that a 164 base pair region of the VP5 promoter gene could activate the transcription of an attached reporter gene (bacteria CAT gene). Furthermore, a 12 bp (GGCCATCTTGAA) cis-acting element situated within the 164 bp promoter region was required for the promoter activity. To understand the function of this cis-element in the regulation of VP5 transcription and to identify the trans-acting factors interacting with this element, gel mobility shift assays were first carried out using the fragment containing the 12 bp site as the probe. A cellular factor, YY 1, was found to bind to this site in a sequence specific manner. Based on the oligonucleotide competition assays, partial protease digestions, and antibody supershift assays, it became clear that two cellular factors bound to the VP5 promoter. These were related, if not identical, to the previously identified Yin-Yang- 1 factor (YY 1), and transcription factor the SPl. Site-directed mutagenesis studies indicated that these two factors bind to distinct sites on the 164 bp fragment. Point mutations studies on the 12 bp YYl binding site demonstrated that seven of the 12 bp were required for YY 1-DNA complex formation and the first four bp in the 12 bp were very important for VP5 gene regulation. Also, it was found that YY 1 performs both positive and negative regulator function in VP5 gene regulation. In conclusion, two cellular transcription factors, YY 1 and SPl, play a major role in VP5 gene expression. |
