HSV-1 ICP4, A Multifaceted RNA PolII Transcription Factor

ICP4, of Herpes Simplex Virus type 1 (HSV-1) is responsible for activation of viral Early and Late genes, and is necessary for viral replication. ICP4 contains two transactivation domains separated by a DNA binding domain. The complex structure of ICP4 indicates the possible diversity of the cellular and viral proteins it interacts with to function. ICP4 interacts with a variety of transcription complexes to promote RNA Polymerase II mediated transcription. The structural basis for these interactions has not yet been clearly defined. To more closely examine the structural requirements for ICP4 activities, mutants in conserved and degenerate regions of the N-terminus, in the presence and absence of the carboxyl terminus, were examined for effects on viral gene expression. It was found that i) the amino terminal transactivation domain is strictly required for E gene transcription, ii) multiple conserved regions within the N-terminus contribute to transcription, and iii) the amino terminal and carboxyl terminal transactivation domains cooperate to mediate transcription. Affinity purification assays demonstrated that many of the observed defects in transcription probably resulted from the deletion of regions involved in stabilizing TFIID. Complementation analyses demonstrated that TFIID interactions are stabilized by the presence of one functional N-terminal and C-terminal transactivation domain within an ICP4 dimer.\ud Affinity purification and mass spectrometry were used to determine the complexity of ICP4 mediated interactions throughout infection in addition to the structural requirements provided by ICP4 for these interactions. Mass spectrometry and western blot data indicated that ICP4 was found in complex with TFIID prior to other components of the transcription machinery including Mediator and TFIIH. Additionally, the amino terminal 774 amino acids were sufficient for interactions with TFIID, Mediator and TFIIH. While ICP4 has previously only been associated with preinitiation complex formation, components of initiation, elongation, mRNA processing, and mRNA export machinery were also found in complexes with ICP4, suggesting that ICP4 functions as a multifaceted RNA PolII transcription factor. Together, the data presented herein provide an understanding of how the structural complexities of ICP4 provide an interface for the formation of transcription complexes. Additionally, a new model for viral transcription is presented. \u