C10: Determining How Activation of the DNA-Dependent Protein Kinase Impacts Adenovirus Replication

While human adenovirus (Ad) infection can cause severe illness, antiviral treatments often cause serious adverse effects and have limited efficacy against severe Ad infection. Ad infection activates the cellular DNA damage response (DDR), which triggers activation of protein complexes such as the DNA-dependent protein kinase (DNA-PK) that hinder viral infection. However, Ad wields several viral proteins from early gene region 4 (E4) that can reduce DNA-PK activation. Previous studies show that Ad mutant viruses lacking E4 (E4-) are replication-deficient but the inhibition of DNA-PK activation by the use of chemical inhibitors can promote the viral life cycle. While DNA-PK activation may impact the viral life cycle, chemical inhibitors can induce off-target effects. We planned to substantiate previous findings derived from using inhibitors by performing siRNA knockdowns of subunits of the DNA-PK complex. We hypothesized that siRNA knockdown of the Ku86 subunit will disrupt DNA-PK activation, allowing us to evaluate how DNA-PK activation impacts the viral life cycle through a more targeted approach. Understanding the mechanisms of DNA-PK suppression of Ad infection can potentially aid in the development of more effective and less toxic antiviral therapies for severe Ad infections in immunocompromised individuals. Thus far our results indicate that our current experimental conditions are sufficient to reduce Ku86 and other cellular protein levels. Moving forward, we plan to investigate if inducing the DDR via DNA damaging agents and E4- virus infection leads to DNA-PK activation after siRNA knockdown of Ku86. Furthermore we would evaluate early protein, late protein, and virion production levels in cells infected with E4- virus after the knockdown of Ku86 to substantiate previous findings. This research project has helped me develop critical skills beneficial for pursuing a research-focused graduate degree in the future.

Author: Cole Pacini, Microbiology

Advisor: Eileen Bridge, Microbiology