For Adenovirus (Ad) to undergo a successful replication cycle, proteins expressed from the early gene region 4 (E4) function in regulating the host’s DNA damage response (DDR). In mutant viruses with a deletion of the E4 region (E4-), one particular DDR kinase — DNA-dependent protein kinase (DNA-PK)— is able to ligate the ends of Ad DNA together forming genome concatemers. It can be predicted the formation of genome concatemers by DNA-PK may have negative effects on the replication cycle of E4- Ad mutants, but not much else is known about the actual effects DNA-PK has on the Ad life cycle. Therefore, we sought to uncover the impacts activation of DNA-PK in E4- infected cells may have on the Ad life cycle in order to better understand the role E4 proteins have on inhibiting the DDR. To test the effects DNA-PK activation has on various stages of the Ad life cycle, a chemical inhibitor was first used to prevent activation of DNA-PK in E4- or wild-type Ad (Ad5) infected cells. Then, several molecular techniques were employed including Western blot analysis to assess levels of viral proteins, qPCR to quantify relative levels of viral DNA, and fluorescent focus assay to measure levels of virion production. Through these techniques we were able to discover that in E4- infected cells, upon addition of the DNA-PK inhibitor, levels of early viral proteins, late viral proteins, and viral yield were significantly increased while DNA replication was not affected. However, when DNA-PK was inhibited in Ad5 infected cells, only early protein levels were increased which may indicate a new role for DNA-PK that is independent of genome concatenation which is completely absent in Ad5 infected cells.
Authors: Meili Aiello, Christopher Chen, and Eileen Bridge
Advisor: Eileen Bridge, Microbiology
Graduate Advisor: Chris Chen, Microbiology
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