Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Microbiology and Immunology

Supervisor

Joseph Mymryk

Abstract

One of the critical functions of human adenovirus (hAd) early region 1A (E1A) protein is to activate transcription of the early viral genes. The largest isoform of E1A contains a unique region termed conserved region 3 (CR3), which includes a Cysteine-4 (C4) zinc finger domain. This region activates viral gene expression by interacting with and recruiting cellular transcription machinery to the regulatory regions of early viral genes. Although this process has been studied at length with hAd type 5 E1A, far less is known about how the E1A proteins from other hAd types activate transcription. There are dramatic differences in the potency of transactivation by E1A CR3s from representative hAd species that cannot be explained by the current model of E1A transactivation. I hypothesized that many of the co-activators targeted by hAd E1A CR3 are conserved between types. However, I also hypothesized that additional cellular factors specific to certain hAd E1A CR3s are also required. The cellular co-activators required by hAd5 E1A CR3 to activate transcription were demonstrated to be conserved among representative members of each hAd species. Furthermore, the cellular lysine acetyl transferase (KAT) GCN5 was identified as a novel negative regulator of E1A CR3 transactivation. The KAT activity of GCN5 was required to exert the effect on E1A CR3. Finally, the C4 zinc finger domain of CR3 is predicted to differ from the rest of E1A by exhibiting a stable structure that is critical for transactivation. A well defined stable solution structure of E1A CR3 was confirmed by NMR spectroscopy. Coordination of a single zinc ion was critical to CR3 structure and folding. Together these observations expand the existing model of E1A CR3 transactivation to not only include representative members of each hAd species, but also implicate a layer of negative regulation and provide structural insight into this paradigm of non-acidic viral transactivators.

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