The expression of herpes simplex virus genes is regulated in a complex and coordinated manner. A major component of the regulatory machinery of the virus is Infected Cell Polypeptide 4 (ICP4). It is absolutely required for virus infection and also for gene expression to proceed past the immediate early phase. ICP4 is a large structurally complex protein, existing in cells as a 350 kd dimer. It can either act as an activator or repressor of transcription. Like many transcriptional regulator proteins, ICP4 is composed of discrete domains specifying a variety of molecular interactions that collectively contribute to its activities. A large part of the proposed studies focuses on the genetic and biochemical definition of the functional domains of ICP4 and how these domains contribute to the regulatory cascade seen in viral infection. A second broad area of the grant deals with reconciling the biochemical activities of ICP4 with its function at different stages of viral infection. The overall approach to these studies involves the construction and analysis of mutant viruses to explore the structure and function of ICP4. Parallel investigations of the biochemical activities of different forms of ICP4 acting on different promoters will provide complementary information.
Five specific aims are proposed to address the long term goals stated above. The ICP4 protein is composed of DNA binding, dimerization and possibly multiple activation domains. In the first aim, experiments are proposed to continue to define the domains of ICP4 that contribute to its regulatory function and to correlate the activity of these domains with function in the context of viral infection. ICP4 is extensively phosphorylated. This modification may affect its ability to bind to DNA, or interact with other proteins involved in the regulatory function of ICP4.
The second aim sets out to determine the sites and consequences of phosphorylation of ICP4.
This aim will involve similar genetic approaches to those used in the previous aim along with phosphotryptic peptide mapping and in vitro transcription to determine how specific modifications of the ICP4 protein affect specific properties or activities. The activity of ICP4 in virus infection may also be augmented and/or altered by the presence of other viral proteins that affect gene expression.
The third aim of the proposal addresses these points through the construction of viruses deleted for defined subsets of selected genes thought to affect or augment ICP4 activity.
The fourth aim of this proposal is to determine the cis and trans acting parameters required for ICP4 to act as a repressor of transcription. This involves investigating the effects on transcription of the relative locations of the TATA box and an ICP4 binding site(s). This information will be interpreted with respect to the ability of ICP4 to form tripartite complexes with TBP (TFIID) and TFIIB, and also with respect to the presence and involvement of upstream sites for cellular and viral activating factors. The last aim is designed to determine the cis and trans acting parameters influencing ICP4 and the discrimination between different classes of HSV promoters.
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