The goal of this research is to define the mechanisms by which the herpes simplex virus ICP27 protein stimulates late viral gene expression through post-transcriptional processes and how HSV infection changes the properties of cellular nuclear proteins such as the retinoblastoma (Rb) gene product and the role of ICP27 in this process. The long term goals of this research are to understand how the ICP27 protein moves into and within the cell nucleus and interacts with specific molecules and structures to effect changes in gene expression and cell metabolism. These proposed studies are of medical relevance because ICP27 provides a potential target for antiviral agents to block HSV replication. In addition, these studies are of general interest in providing a system to study how a virus inhibits the progression of the cell cycle and to study possible mechanisms for controlling cell growth. First, the effect of ICP27 on late gene expression will be studied by analyzing late RNA transport, processing and stability in wild type and ICP27 mutant virus-infected cells. Ribonucleoprotein complexes formed with late RNAs in infected cells and in vitro with infected cell extracts will be examined to determine whether changes in these complexes can explain the effect of ICP27 on post- transcriptional steps in late gene expression. Second, an approach will be developed using in situ hybridization with cultured cells for determining the intranuclear location of late viral transcripts, and this will be used to determine how the intranuclear location of late viral transcripts changes during viral infection and how ICP27 might affect this parameter of viral gene expression. Third, the effects of HSV infection and of ICP27 on the properties of the retinoblastinoma gene product (Rb) will be determined by defining the biochemical basis for the change in the forms of Rb in HSV infected cells, defining the mechanism of the requirement for ICP27 for this effect, and determination if ICP27 is sufficient to mediate this effect. Fourth, the amino acid signals on ICP27 which target it to the cell nucleus will be examined by constructing gene fusions between the genes for ICP27 and a cytoplasmic protein, pyruvate kinase.
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