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CHARACTERIZING LOCALIZATION OF ADENOVIRUS PROTEIN E4 11K AND THE CELLULAR DOUBLE-STRAND BREAK MODULATOR DNA-PK

Abstract

Adenovirus (Ad) is a linear, double-stranded DNA virus that causes a variety of mild infections. The Ad genome can be broken up into smaller regions that encode proteins necessary for successful viral replication. Our protein of interest, E4 11k, is encoded by the “early” region of the Ad genome and has important functions in inhibiting normal host cell processes, such as host cell antiviral signaling pathways. Additionally, E4 11k is suspected to have an important role in the inhibition of the non-homologous end joining (NHEJ) pathway of double strand break (DSB) DNA repair. During NHEJ, DNA-dependent protein kinase (DNA-PK), initiates DNA ligation through autophosphorylation. This pathway is triggered by the presence of double-stranded DNA (dsDNA) break products, or during an adenoviral infection, due to the viral genome’s similarity to a dsDNA break. However, success of this pathway is not seen during wild-type adenovirus infections, indicating adenovirus has evolved some way to evade this pathway. Past research done on activation levels of DNA-PK in the presence of adenoviral mutants indicates that the presence of E4 11k is necessary to inhibit phosphorylation of DNA-PK. Our research aims to use immunofluorescence to determine the individual efficiency of E4 11k to inhibit DNA-PK phosphorylation. A549 lung carcinoma cells were infected with adenovirus mutants and/or treated with dsDNA break inducer etoposide before protein staining and slide fixation. Proteins of choice were then tagged using fluorescent antibodies and visualized using confocal microscopy. Initially, the localization of E4 11k, phosphorylated DNA-PK, and total DNA-PK were visualized. Additional staining of cytoskeletal protein gamma-tubulin, known E4 11k binding partner promyelocytic leukemia protein, and viral replication marker DNA-binding protein were performed. Protein localization staining was completed for in mock-infected, wild-type Ad-infected, E4 11k-only-infected, and dsDNA break-induced cells. While past research suggests that E4 11k is necessary for inhibition, initial findings show no consistent indication that E4 11k has any role in directly inhibiting DNA-PK phosphorylation.

Acknowledgements

GCSU MURACE 2025 Summer Research Grant, GC Journey's Mini Grant, Academic Affairs Small Grant, Tri-Beta Undergraduate Research Grant

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