Defense Date
6-3-2024
Graduation Date
Summer 8-2024
Availability
Immediate Access
Submission Type
dissertation
Degree Name
PhD
Department
Biological Sciences
School
School of Science and Engineering
Committee Chair
Jill Dembowski
Committee Member
Nancy Trun
Committee Member
Lauren O'Donnell
Committee Member
Michael Jensen-Seaman
Keywords
HSV-1, virus, viral infection, DNA replication, DNA, herpes simplex virus type 1, PCNA, proliferating cell nuclear antigen
Abstract
Herpes simplex virus type 1 (HSV-1) is a double stranded DNA virus that replicates within the nucleus of host cells. Using accelerated native isolation of proteins on nascent DNA (aniPOND), cellular proteins were found to associate with HSV-1 replication forks. One host factor of interest is proliferating cell nuclear antigen (PCNA), the DNA sliding clamp that tethers DNA polymerases and DNA damage response proteins to replicating cellular DNA. Although HSV-1 encodes its own DNA polymerase processivity factor (UL42), PCNA plays a key role in HSV-1 infection. We used commercially available PCNA inhibitors and short hairpin RNAs (shRNA) to investigate how HSV-1 modifies, recruits, and utilizes PCNA. We found that PCNA inhibition results in decreased viral yield, a reduction in viral late mRNA and protein expression, and inhibits the association of select viral and cellular factors with replicating HSV-1 DNA. We also found by immunoprecipitation that PCNA copurifies with viral capsid, structural, and tegument proteins, specifically the viral deubiquitinase, UL36. We determined that PCNA ubiquitination peaks at 4 hpi and then decreases after the onset of viral DNA replication and late gene expression, perhaps due to UL36 activity. Finally, we used inducible shRNAs that target PCNA. We found that PCNA knockdown at 96-hours post doxycycline induction results in a 10-fold reduction in viral yield. These data reveal an important role for PCNA at viral replication forks in vivo, demonstrate that PCNA is modified during infection, and that PCNA inhibitors have the potential to be adapted for antiviral treatment against HSV-1 infection.
Language
English
Recommended Citation
Packard, J., & Dembowski, J. A. (2024). Manipulation of Cellular Proliferating Cell Nuclear Antigen (PCNA) During Herpes Simplex Virus Type 1 Infection (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/2305
Additional Citations
Packard JE, Williams MR, Fromuth DP, Dembowski JA. Proliferating cell nuclear antigen inhibitors block distinct stages of herpes simplex virus infection. PLoS Pathog. 2023;19(7):e1011539. Published 2023 Jul 24. doi:10.1371/journal.ppat.1011539
Packard JE, Dembowski JA. HSV-1 DNA Replication-Coordinated Regulation by Viral and Cellular Factors. Viruses. 2021;13(10):2015. Published 2021 Oct 7. doi:10.3390/v13102015
Packard JE, Kumar N, Weitzman MD, Dembowski JA. Identifying Protein Interactions with Viral DNA Genomes during Virus Infection. Viruses. 2024;16(6):845. Published 2024 May 25. doi:10.3390/v16060845
