Identification and characterization of protein interaction domains in the herpes simplex virus type I transcription factor ICP4

Herpes Simplex Virus type I (HSV-1) is an important human pathogen. The 70 genes of the virus are transcribed by the host RNA polymerase II in three kinetic classes: immediate early, early, and late. One of the immediate early proteins, infected cell polypeptide 4 (ICP4) is an essential transcription factor that regulates viral gene expression. The focus of this dissertation research was to identify the regions of ICP4 that are required for regulation of transcription and to investigate the mechanisms by which ICP4 affects transcription.; The first goal of my research was to identify domains in the C-terminus of ICP4 that are required for activation of viral promoters. Using a transient transfection assay to analyze a series of ICP4 variants with C-terminal truncations or point mutations, we observed that residues 1252–1254 specifically affected the activation activity of ICP4 but had no effect on the repression activity of ICP4.; One of the proteins we identified as binding to ICP4 was casein kinase 2 (CK2). Variants of ICP4 that encode in-frame deletions of residues 176–206 repress transcription 2–4 fold more than WT ICP4. This increased repression activity by ICP4 ΔSEAC variants correlates with the loss of binding to CK2. We propose that either CK2 binding or phosphorylation of ICP4 residues 176–206 decreases the repression activity of ICP4.; The data presented in this investigation are consistent with the function of ICP4 as either a co-activator or derepressor of viral gene expression. Residues between 1243–1298 encode a function that contributes to activation of transcription by ICP4. If ICP4 functions as a co-activator, ICP4 residues 1243–1298 may bind directly to, or stabilize a domain of ICP4 required to bind to, a cellular protein that activates transcription. If ICP4 acts as a derepressor, ICP4 may enzymatically modify, inhibit or displace a repressive proteins (such as histones) from viral promoters. ICP4 residues 1243–1298 may directly or indirectly interact with the repressive proteins. ICP4 is a large viral regulatory protein that potentially interacts with many cellular proteins; characterization of these interactions are important for understanding the complex life cycle of HSV and treating HSV infections. (Abstract shortened by UMI.)...