The purpose of the proposed investigation is to purify and characterize the viral polypeptide designated ICP4 which is synthesized within two hours after productive infection of animal cells by herpes simplex virus. Results from other investigations suggest that ICP4 functions as a regulatory protein to control the transcription of viral genes exposed during the delayed early phase of productive infection. In this investigation, two approaches will be used to elucidate the molecular processes which act to control and coordinate gene expression in HSV-infected cells. The first approach will require the purification of ICP4 in a native, soluble form. To maximize the yield of ICP4 from HSV-infected cells, conditions for overproduction of ICP4 will be established. To simplify the purification process, a rapid assay for ICP4 employing immunological techniques will be developed. Copurification of other polypeptides with ICP4 will be monitored to examine the possibility that ICP4 functions within a multimeric complex rather than as a monomeric protein. The purified protein will be characterized with respect to both structural and functional properties that could be relevant to a mechanism for transcriptional control. Specifically, the affinity of the purified protein for defined regions of HSV DNA will be determined and the effect of the addition of ICP4 to in vitro transcription systems employing HSV DNA fragments as templates will be monitored. The second approach to understanding the function of ICP4 will focus on elucidating the association of ICP4 with other nuclear components in situ. Nuclear complexes containing ICP4 will be immunoprecipitated with anti-ICP4 IgG; the components of the complexes will be analyzed for the presence of specific viral and/or host proteins and specific regions of viral DNA. Changes in the components of the immunoprecipitated complexes during the viral reproductive cycle will be monitored. The combined results from these two approaches will be used to propose a model for the regulation of viral delayed early gene expression by ICP4 in HSV-infected cells.
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