The herpes simplex virus (HSV) thymidine kinase (tk) gene provides an excellent model system for studies of the regulation of eukaryotic gene expression, particularly in HSV-infected cells. Such studies are especially health-related: HSV and other herpes viruses cause diseases for which there are treatments such as acyclovir (ACV) but no cure. The tk gene is a site for ACV-resistance, which is a growing problem in immunocompromised patients. The general aim of this project is to understand mechanisms pertinent to HSV tk expression, especially from the viral genome during infection. The first specific aim addresses the hypothesis that HSV immediate early regulatory proteins, specifically ICP4, induce tk expression via the cellular transcription factor, TFIID. The tk TATA box will be substituted with """"""""wild-type"""""""" TATA boxes known to respond differently to different regulatory proteins. The substitutions will be introduced into an ICP4-deficient virus, permitting examination of their effects in the presence or absence of ICP4. The second specific aim focuses on a transcription signal downstream of the tk MRNA cap site. The transcription factor that binds this signal will be identified using standard procedures such as mobility shift assays, and the sequence requirements for binding will be compared with the importance of these sequences for tk transcription. Additionally, the effects of this signal and other mutations on the time course of tk expression in the presence or absence of DNA replication inhibitors will be tested. The third specific aim will determine if tk transcripts regulate the expression of an overlapping gene, UL24, by antisense mechanisms by assaying the effect of mutations that reduce tk expression on UL24 transcript levels and sequence modifications, and on the synthesis of UL24 protein. Should antisense regulation be found, the effect of overexpressing UL24 on HSV infection will be tested. The fourth specific aim seeks to determine whether frameshifting occurs during translation of tk MRNA containing a single base insertion, such as one expressed by an acyclovir-resistant clinical isolate. The sequences controlling such frameshifting will be mapped using in vitro transcription and translation methods.
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