The overall goal of this study is the elucidation of the molecular interactions and events underlying the cascade of herpes simplex virus (HSV-1) transcription during productive infection. Despite knowing of the existence of this regulatory cascade for 4 decades, our knowledge of the molecular underpinning for it is strikingly lacking. Detailed knowledge of how HSV genes are transcribed, and the virus-cell interactions that contribute to their regulated expression, may allow for strategies to block activated transcription and hence virus multiplication. The viral genome is the only component of the virus present throughout its productive life cycle. However, the viral and cellular proteins that bind to, and th processes that occur on the genome change as the life cycle progresses from entry to the packaging of viral genomes into nascent virions. We have developed an approach, which is a modification of a technique called isolation of proteins on nascent DNA (iPOND) to determine in a nonbiased way the viral and cellular proteins that associate with the genome throughout infection, from the initial sensing of the genome to its packaging. Of relevance to this proposal are the viral and cellular proteins involved in RNA polymerase II transcription that associate with the genome, as infection proceeds. Thus, the first aim is to interrogate the viral and cellular proteins that bind to the genome throughout infection and differentiate between those binding at replication forks and those binding to replicated DNA. The viral Infected Cell Polypeptide 4 (ICP4) is large multifunctional transcription regulator that binds to the viral genome. The cellula proteins/complexes that affinity-purify with ICP4 change as infection proceeds, and are involved in all stages of transcription. The most abundant of these are the most fundamental of general Pol II transcription factors, TFIID and mediator. Mediator(s) are variable multicomponent complexes that can positively and negatively affect the rates of transcription initiation and elongation. ICP4 may interact with multiple components/forms of mediator to differentially regulate viral genes.
The second aim determines the forms of mediator that interact with ICP4 and the genome and to test the hypothesis these interactions contribute to activation and turn off of different HSV gene in the regulatory cascade. Multiple chromatin remodeling complexes were found on the viral genomes, some of which interact with ICP4. Histones appeared to be excluded, while ICP4 is one of the most abundant proteins on the genome. Preliminary ChIP-Seq experiments show that as DNA replication proceeds, the density of ICP4 binding is reduced and Pol II occupation of late genes is increased.
The third aim addresses the hypothesis that ICP4 is both a mediator of cellular chromatin, helping to keep transcribing and replicating DNA free of histones, and that it also serves as a type of viral chromatin to regulate the expression o late genes. Together these studies will address the understudied more dynamic aspects of the regulatory cascade of HSV transcription.

Public Health Relevance

The overall goal of this study is the elucidation of the molecular interactions and events underlying the cascade of herpes simplex virus (HSV-1) transcription during productive infection, following the activation of Immediate Early (IE) genes. Detailed knowledge of how HSV genes are transcribed, and the virus-cell interactions that contribute to their regulated expression, may allow for strategies to block activated transcription and hence virus multiplication.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI030612-24
Application #
9601621
Study Section
Virology - A Study Section (VIRA)
Program Officer
Natarajan, Ramya
Project Start
1992-03-01
Project End
2020-11-30
Budget Start
2018-12-01
Budget End
2019-11-30
Support Year
24
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Genetics
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Dembowski, Jill A; DeLuca, Neal A (2018) Temporal Viral Genome-Protein Interactions Define Distinct Stages of Productive Herpesviral Infection. MBio 9:
Fox, Hannah L; Dembowski, Jill A; DeLuca, Neal A (2017) A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts. MBio 8:
Dembowski, Jill A; Dremel, Sarah E; DeLuca, Neal A (2017) Replication-Coupled Recruitment of Viral and Cellular Factors to Herpes Simplex Virus Type 1 Replication Forks for the Maintenance and Expression of Viral Genomes. PLoS Pathog 13:e1006166
Dembowski, Jill A; Deluca, Neal A (2017) Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins. J Vis Exp :
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Wagner, Lauren M; DeLuca, Neal A (2013) Temporal association of herpes simplex virus ICP4 with cellular complexes functioning at multiple steps in PolII transcription. PLoS One 8:e78242

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