The long term goals of this research are to understand how the herpes simplex virus (HSV)-encoded transcriptional activator protein, ICP4, moves into and within the cell nucleus and interacts with specific structures and molecules in the nucleus to effect a change in transcription. This proposal focuses on the mechanism of nuclear localization of ICP4, the interaction of ICP4 with cellular proteins in the nucleus, the defects in localization of a dominant temperature sensitive mutant ICP4, and the nature of an ICP4-related antigen expressed on the infected cell surface. The mechanism of nuclear localization will be studied by determining the ICP4 nuclear localization signals using gene fusions and in vitro mutagenesis. The pathway of localization of wild type and mutant ICP4 will be studied by cell fractionation and biochemical analysis of potential transport intermediates. To examine interactions of ICP4 with host cell molecules, apparent interactions between ICP4 and nuclear actin will be examined using immunoprecipitation techniques. The distribution and form of nuclear actin will be studied in normal and HSV- infected cells by immunofluorescence and cell biological techniques that define the state (globular vs. filamentous) of actin and its intracellular location. The metabolism of nuclear actin will be examined by two-dimensional protein gels and in vitro nuclear transcription assays to measure the rates of synthesis of the different actin isotypes in normal and HSV-infected cells. Using transient expression assays, it will be determined whether the mutant ICP4 protein can inhibit nuclear localization of cellular and other viral proteins. This could define nuclear proteins that share pathways of localization. The identity of an ICP4-related protein expressed on the cell surface will be determined by surface labelling and immunoprecipitation techniques. The function of this surface antigen will be explored by examining viral gene expression in the presence of antibody and isolating viral mutants that do not express the surface antigen. These studies may yield information about mechanisms of HSV latency in that the ICP4 gene function may be important in the control of viral replication during establishment or maintenance of HSV latent infection. These studies may also yield basic information about mechanisms of transcriptional activation and the functions of nuclear actin.
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