The long-term objective of this application is to study novel post-transcriptional regulatory mechanisms used by the herpes simplex virus (HSV). A focus of these studies is the HSV thymidine kinase (tk) gene and the gene it overlaps, UL24. HSV and other herpesviruses are important human pathogens. TK mediates antiviral drug sensitivity and drug resistance, which is an important clinical problem in patients such as those with AIDS. The first specific aim explores the kinetics of UL24 expression and its regulation by HSV ICP27. The hypothesis that different (short and long) UL24 transcripts exhibit different kinetics due to their different poly(A) signals will be analyzed by constructing recombinant viruses containing these signals and a """"""""leaky"""""""" late promoter in the tk gene, using bacterial artificial chromosome (BAC) technology. Protein factors that interact with these signals will be identified. The sequences important for differential regulation of UL24 transcript abundance and localization by ICP27 will also be studied with the use of recombinant viruses. The hypothesis that this regulation is due to a role for ICP27 in transcriptional elongation will be tested collaboratively. The biological role of the different UL24 transcripts will be investigated by construction of viral mutants that express only these transcripts or only the long transcripts. The second specific aim analyzes the mechanism and consequences of an unusual internal ribosome entry sequence (IRES) in tk mRNA. How this short sequence that lacks an AUG interacts with the translation machinery and positions ribosomes for initiation will be assessed in vitro. Expression from the IRES will be examined in virus-infected cells and how it promotes TK activity will be investigated using E. coli expressed TK polypeptides. The third specific aim starts from the recent identification of HSV-encoded microRNAs (miRNAs). HSV-encoded miRNAs will be catalogued, precisely mapped, and their biosynthesis investigated with the aid of molecular genetic and pharmacological tools. Viral and cellular targets for the miRNAs will be predicted and tested, and the mechanisms and consequences of their post-transcriptional actions will be explored. The relevance of these three aims for public health is that they explore gene regulatory mechanisms important for disease and for resistance of pathogens to antiviral drugs.
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