The long-term objective of this application is to study post-transcriptional regulation of the herpes simplex virus (HSV) thymidine kinase (tk) gene and the gene it overlaps, UL24. This locus provides an excellent model system for studies of post-transcriptional events, particularly in HSV- infected cells. HSV and other herpesviruses are important human pathogens. TK mediates anti-viral drug sensitivity and drug resistance, which is an important clinical problem in patients such as those with AIDS. The first specific aim explores the regulation of UL24 expression and other HSV genes by polyadenylation. The poly(A) signals will be analyzed by constructing recombinant viruses and in extracts of cells. Protein factors that interact with these signals will be identified. The role of HSV ICP27, which acts after transcriptional initiation in the synthesis of certain UL24 transcripts, will be studied with the use of recombinant viruses also. The second specific aim investigates antisense regulation of UL24 by tk, which may entail novel mechanisms. The presence of tk- UL24, double-stranded RNAs and the effects of tk expression on stability, localization, modifications, and translation of UL24 transcripts will be explored as will the ribosomal frameshifting of tk mRNA from acyclovir- resistant clinical isolates. For one such isolate, the frameshift sift will be sequenced and the roles of non-Watson-Crick base pairs and potential interactions with ribosomal RNA in this unusual frameshift will be explored. For at least three other tk mutants, studies will be initiated to determine if frameshifting occurs to permit expression of TK and whether the mechanisms involved are novel. These studies link the molecular biology of frameshifting with the clinical problem of drug resistance.
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