Fernandez, Rosio
(2024)
Determining the interactions between Mediator, ICP4 and the HSV-1 genome.
Master's Thesis, University of Pittsburgh.
(Unpublished)
Abstract
Herpes Simplex Virus-1 (HSV-1) is a large double stranded DNA (dsDNA) virus that infects the oral and genital mucosa resulting in a lifelong incurable infection persisting latently in the innervating sensory ganglia. The 152 kilobase dsDNA virus employs a coordinated cascade to sequentially transcribe over 80 genes to efficiently generate viral progeny to high titers, which can induce severe pathologies including encephalitis, keratitis, and pneumonia in immunocompromised individuals. The HSV-1 genome is transcribed in the nucleus and co-opts the host RNA Polymerase II (Pol II) transcriptional machinery. Our lab has demonstrated that the viral protein, Infected Cell Polypeptide 4 (ICP4), is required for this reprogramming of host transcription. ICP4 is a dsDNA binding protein that recruits host transcription factors—TFIID, and Mediator—to the viral genome. Specifically, we wanted to determine the specific interactions between ICP4 and Mediator to discern the role that Mediator plays in the temporal regulation of HSV-1 transcription. This involves determining which regions of ICP4 interacts with which subunits of Mediator, and how these interactions affect the recruitment of Mediators to the viral genome, and subsequently viral transcription and growth. The interactions between ICP4 and Mediator, and Mediator and the HSV genome are likely to be complex, with the forms or composition of mediator interacting with ICP4 and the viral genome potentially changing throughout infection to facilitate elements of the HSV transcription cascade. Advancing this may aid in distinguishing a unique viral-host interaction for novel drug targets and development of therapeutics.
Mediator is an evolutionarily conserved multiprotein protein complex comprised of at least 30 variable subunits. Mediator acts as a functional bridge between transcription factors and the basal transcriptional machinery on core promoters to facilitate in the activation, repression, and elongation of cellular transcripts.
To expand on these observations, we tried to employ a proximity-based labeling assay to determine the variable composition of Mediator associated with ICP4 during infection and Tandem affinity purification (TAP) of ICP4-associated cellular proteins with intact ICP4 and established ICP4 mutant viruses targeting various regions of the protein.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
| Title | Member | Email Address | Pitt Username | ORCID |
|---|
| Committee Chair | Lee, Nara | | | | | Thesis Advisor | DeLuca, Neal | ndeluca@pitt.edu | | | | Committee Member | Flynn, JoAnne | | | | | Committee Member | Schmidt, Martin | | | | | Committee Member | Bomberger, Jennifer | | | |
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| Date: |
26 June 2024 |
| Defense Date: |
1 July 2024 |
| Approval Date: |
14 October 2024 |
| Submission Date: |
16 July 2024 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
57 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Microbiology and Immunology |
| Degree: |
MS - Master of Science |
| Thesis Type: |
Master's Thesis |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
HSV-1, Transcription, ICP4, Mediator |
| Date Deposited: |
14 Oct 2024 14:49 |
| Last Modified: |
14 Oct 2024 14:49 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/46692 |
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