Adenovirus (Ad) infection can lead to the activation of DNA damage response (DDR) pathways such as non-homologous end joining (NHEJ) if unchecked. To avert DDR interference, Ad encodes several proteins in early region 4 (E4) that regulate the DDR to promote viral replication. Specifically, E4 11kDa and 34kDa have been shown to interact with DNA-dependent protein kinase (DNA-PK), a key NHEJ mediator, disrupting NHEJ. Though E4 protein interactions with DNA-PK have been shown, several decades have passed since the molecular mechanism by which E4 proteins regulate DNA-PK has been investigated. Furthermore, it is not clear whether regulatory functions of the E4 proteins are redundant or interdependent. The goals of this study were to further the fields of adenovirus and microbiology by 1) shedding light on how the viral E4 11kDa and E4 34kDa proteins regulate DNA-PK during infection and 2) whether the E4 proteins are interdependent. To address our study goals, we used Ad mutants containing deletions of E4 11kDa or 34kDa to infect cervical cancer (HeLa) cells and analyzed the profile of DNA-PK activation during infection. To assess how levels of DNA-PK activation were altered, we used western blot analysis. We also used immunofluorescence microscopy to elucidate if the presence of either E4 protein affected the cellular localization of DNA-PK. Preliminary data gathered from our study indicates that either E4 protein can reduce the levels of activated DNA-PK during infection. These findings suggest that both E4 11kDa and 34kDa may redundantly regulate DNA-PK. Moving forward, we intend to clarify these findings by transfecting HeLa cells with plasmids encoding E4 11kDa or 34kDa and evaluating effects of their expression on DNA-PK activation and localization outside of viral infection. This work has provided me with invaluable experience working with mammalian cell culture, foundational in my aspired career field of virology.
Author: Cole Pacini
Faculty Advisor: Eileen Bridge, Microbiology
Graduate Student Advisor: Christopher Chen
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