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CHARACTERIZATION OF THE INTERACTION BETWEEN ADENOVIRAL PROTEIN E4 11K AND THE DNA-PK-DEPENDENT DNA DAMAGE RESPONSE**

Abstract

Adenovirus (Ad) can cause upper respiratory infections, gastroenteritis, and conjunctivitis. The linear, double-stranded DNA Ad viral genome can be broken up into early and late phase genes. Our protein of interest, E4 11k, is a product of one of the early genes that works to disrupt normal cellular function to enhance the viral replication cycle. One of the mechanisms E4 11k uses to achieve this disruption is through the inhibition of the DNA damage response (DDR). The DDR is responsible for repairing double-stranded DNA breaks (DSBs) through non-homologous end joining (NHEJ). During DDR, DNA-dependent protein kinase (DNA-PK) becomes activated via phosphorylation. During an infection, Ad’s genome triggers the DSB repair pathway due to the genome’s structural similarity to a cellular DSB. The DDR tries to “repair” the viral genome by ligating the viral genomes into concatemers, disrupting viral DNA replication. Ad, however, has evolved mechanisms to prevent activation of the DDR. Previously, E4 proteins have been shown to regulate the activation of DNA-PK. In this study, we aim to determine E4 11k’s ability to inhibit phosphorylation of DNA-PK in response to cellular DSBs. HeLa (cervical cancer) cells were treated chemically with different combinations of either etoposide (drug that induces DSBs), Nu771 (inhibitor of DNA-PK phosphorylation), or an Ad E4 11k only viral infection. The phosphorylation status of DNA-PK will be analyzed through immunoblotting. In the future, we want to characterize DNA-PK localization during an Ad infection. Immunofluorescence microscopy will be used to visualize protein localization. Our initial findings indicate a 97% reduction in DNA-PK activation. It has previously been shown that etoposide treatment combined with the inhibition of DNA-PK activation result in cancer cells that have less adherence. These findings suggest a potential mechanism for which E4 11k can be used as a novel chemosensitizer for therapy-resistant cancer cells.

Acknowledgements

John E. Sallstrom Honors College Transformative Experience Grant, GC Journey's Mini Grant, Academic Affairs Small Grant, Saladin Scholarship, & Tri-Beta Undergraduate Research Grant

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