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The research proposal centers on two on going projects. The first project rests on the previous established evidence that the herpes simplex virus 1 (HSV-1) genome consists of two covalently linked components consisting of unique sequences flanked by inverted repeats. The two components invert relative to each other and that the viral DNA consists of 4 isomers differing in the orientation of the two components. Previous studies based on the construction of novel genomes established that the cis-acting inversion site resides in a 500 bp a sequence, that inversions are not essential for virus growth inasmuch as the entire inverted repeat sequence can be deleted, and that the inversions are mediated by as yet unidentified trans-acting viral factors. The research objectives are as follows: (i) to identify the cis-acting sites mediating the inversions, (ii) to identify the viral trans-acting factors involved in the inversions, (iii) to investigate the significance of the accruing evidence that the 4 isomers may not be functionally evident, and (iv) to determine the biologic function of the internal inverted repeats. The projected studies entail construction of novel genomes to identify the cis-acting site and the biologic properties of the 4 isomers and of the internal inverted repeats. Both biochemical and genetic studies will be done to identify the trans-acting factors. Project 2 is based on studies showing that translation of viral mRNA is in certain instances regulated at the cytoplasmic level. We have selected the glycoprotein B gene in order to establish the nature of the cis-acting and, possibly, trans-acting factors involved in the translational regulation defines by the presence in the cytosol of competent viral mRNA that us not translated, The experimental design entails construction of novel viral genomes in which (i) the promoter regulatory domain is replaced, (ii) the 5' transcribed, non coding sequences are replaced and (iii) a second copy of the gene is inserted into the genome at another site and this domain of that copy is deleted. In each instance, we will assay the kinetics of accumulation of viral protein in the infected cell and of competent mRNS by in vitro translation. The objective is to identify the cis-acting sequence and/or the transacting factors that turn off translation notwithstanding continued transcription and accumulation of competent mRNA in the cytoplasm.
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