Infection by herpes simplex virus type 1 (HSV-1) can result in relatively mild cutaneous lesions, and more severe outcomes such as blindness and encephalitis. These diseases are the result of expressed viral gene products during the productive replication cycle of the virus and the host's response to infection. The ~80 viral genes are expressed in an ordered cascade of three general classes;immediate early (IE), early (E) and late (L) genes. Each gene has its own promoter, and the architecture of each class of promoter is distinct. However, details regarding the requirements for expression of the three classes of genes are not entirely understood. ICP4 is a transactivator of polII transcription that is required for the efficient expression of viral early and late genes, and hence viral growth. In eukaryotic cells, different core promoter recognition factors/complexes may be involved in regulating the transcription of cellular genes with different core promoter architectures. We hypothesize that ICP4 interacts with different cellular transcription factors, including different core promoter recognition factors, during different stages of infection to differentially activate early and late genes, thereby contributing to the observed regulatory cascade. Data is presented that the cellular complexes TFIID and med are be two such factors. It also follows that distinct but also overlapping domains of ICP4 are involved in the interactions resulting in the activation of early and late genes. Three interwoven specific aims are proposed to address these hypotheses: (i) Determine and characterize the ICP4-containing complexes seen in HSV infected cells by affinity purification and proteomic approaches, and ascertain how they quantitatively and/or qualitatively change throughout infection. (ii) Virus genetics experiments will be conducted to determine the regions of ICP4 required for the formation of the complexes indicated in aim 1, and the consequence of these regions (and hence the interactions) for viral gene expression during infection. (iii) Chromatin immunoprecipitation (ChIP) experiments will be conducted on infected cells to determine how the complexes discovered in aim 1 associate with the different classes of promoters and affect the assembly of transcription initiation complexes throughout infection. Mutants defective in the interactions discovered in aim 1 (from aim 2) will also be analyzed by ChIP to determine how the interactions affect transcription complex formation on HSV promoters. Outcomes will be interpreted with respect to the gene expression phenotypes of the mutant viruses. All of the proposed genetic and biochemical experiments are conducted in the physiologically relevant context of viral infection and are designed to elucidate molecular details of the regulation of HSV gene transcription and eukaryotic transcription program switching in general. The approaches also account for the changes in the cellular transcriptional machinery that may occur as infection proceeds. Knowledge of these molecular details should eventually translate to strategies to control events in the virus life cycle.

Public Health Relevance

The diseases caused by HSV are the result of expressed viral gene products during the lytic replication cycle of the virus, and the host's response to infection. The focus of this competitive renewal application is to determine the viral and cellular proteins and mechanisms that contribute to the regulated transcription of HSV genes. Detailed knowledge of how HSV genes are transcribed and what interactions contribute to their regulated expression may allow for strategies to block activated transcription and hence virus multiplication.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Special Emphasis Panel (ZRG1-IDM-Q (02))
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Challberg, Mark D
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University of Pittsburgh
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Dembowski, Jill A; DeLuca, Neal A (2018) Temporal Viral Genome-Protein Interactions Define Distinct Stages of Productive Herpesviral Infection. MBio 9:
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