Browsing by Author "Katzenellenbogen, Rachel"

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    Conserved Residues in Murine Papillomavirus E2 Regulate the Viral Life Cycle
    (2024-12) Gonzalez, Jessica Kay; Androphy, Elliot J.; Dong, X. Charlie; Katzenellenbogen, Rachel; Robinson, Christopher
    Papillomaviruses (PVs) are small, non-enveloped DNA viruses that infect the stratified epithelia. Once an infection is initiated, the virus must successfully navigate the three stages of its life cycle: establishment, maintenance, and vegetative amplification. A major mechanism of regulating this viral program is post-translational modification on the viral E2 protein, which is responsible for orchestrating viral transcription, replication, and genome partitioning. The hypothesis underscoring this work is that residues in E2 are highly conserved across PV types because they serve some structural or functional purpose for the virus. A targeted mutant library was generated in E2 from murine papillomavirus (MmuPV1) to investigate conserved residues that have been shown to be post-translationally modified in the E2 of other PVs, including BPV-1 and high-risk HPV-31. In the transactivation domain (TAD) tyrosine 102 and the lysine 112/113 motif were modified to their constitutively modified (phosphorylated and acetylated, respectively) or unmodified states, while cysteine 307 in the DNA binding and dimerization domain (DBD) was mutated to a less-reactive serine or DNA binding defective phenylalanine mutant. We characterized how mutation at each of these conserved sites alters E2 function using a battery of in vitro assays to assess for transcription and replication ability. We also studied how each mutant contributes to disease progression using an immunocompromised mouse model assessing cutaneous disease. We demonstrate that mutants which fail to replicate transiently in vitro will also fail to induce proliferative wart formation, establishing a predictive link between in vitro and in vivo experiments. Taken together, our findings suggest that modifications on conserved residues in E2 act as molecular switches that regulate E2 activity throughout the cellular and viral life cycle.
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    Longitudinal Keratinocyte Proliferation Induced by Human Papillomavirus 16E6 and NFX1-123 Partnership
    (2025-04) Billingsley, Caylin Leann; Katzenellenbogen, Rachel; Androphy, Elliot; Mosley, Amber; Robinson, Christopher
    High-risk human papillomaviruses (HPV) cause cervical, anogenital, and head and neck cancers. Despite the availability of preventive HPV vaccines, their poor uptake leaves most men and women at risk for these cancers, many of which remain common globally and others that are increasing domestically. The HPV 16 E6 (16E6) protein plays a significant role in inducing and maintaining cellular transformation of its infected host cell; however, 16E6 itself has no enzymatic activity and carries out most of its functions through partnerships with host endogenous proteins. Previously, it was demonstrated that 16E6 binds to the host protein NFX1-123, and NFX1-123 expression is increased in HPV 16 positive cervical cancer cell lines and primary cancers compared to normal tissues. In this thesis, we quantify the growth rates of 16E6-expressing keratinocytes with endogenous or overexpressed NFX1-123 (16E6/vec and 16E6/FN123, respectively) levels by measuring population doublings and interrogate whether proteome expression changes occur early or late in longitudinal growth assays that began with higher levels of NFX1-123. Early passage 16E6/vec and 16E6/FN123 cells showed similar growth rates; however, late passage 16E6/FN123 cells had accelerated growth and greater population doublings than the 16E6/vec cells. Mass spectrometry revealed similar proteomes of both cell lines at early passages. In contrast, late passaged cells had significantly higher amounts of differentially expressed protein among 16E6/FN123 and 16E6/vec cells. This indicates a unique proteomic landscape induced by the 16E6/NFX1-123 partnership. Pathway analysis vii showed increased positive telomere regulation, DNA repair, and DNA replication pathways in the late passage 16E6/FN123 cells compared to 16E6/vec. These findings indicate that the 16E6 and NFX1-123 partnership, especially with higher levels of NFX1-123, alters the longitudinal cellular environment in a manner that may initiate a preneoplastic phenotype. Additionally, the work detailed in this dissertation provides the first evidence of NFX1-123 being a direct RNA-binding protein giving new insights into the endogenous roles of the protein.