Characterizing the C-terminal Region of Human Adenovirus E1A: An Undiscovered Country

Human Adenovirus (HAdV) E1A is the first protein expressed during viral infection. The primary function of E1A is to reprogram the cell for viral replication, but it is additionally capable of transforming primary rodent cells in co-operation with other oncogenes such as HAdV E1B. Despite extensive study, little is known about the function and cellular targets of the C-terminal region of E1A. Importantly, this region is required for the transforming ability of E1A with E1B, but can also suppress transformation with Ras. Previous studies showed that interaction with the C-terminal Binding Protein (CtBP) plays a role in both functions described above. However, other factors must be necessary, as there are mutants of E1A that retain CtBP binding but fail to contribute to either effect. Given the recent identification of new targets of this region of E1A, including FOXK1/2, DYRK1A/1B, and HAN11, I sought to re-evaluate and further characterize the mechanism by which the C-terminus of E1A carries out its functions. I performed an extensive and systematic mutational analysis of the C-terminus of E1A as a means of identifying residues specifically required for binding each cellular target. We then tested our panel of mutants for their ability to transform primary baby rat kidney cells in cooperation with E1B or Ras. Contrary to the current understanding of how the C-terminus of E1A performs its functions, my findings indicate that while CtBP binding is required for transformation with E1B, it is not necessary for the suppression of transformation with Ras. This suggests that other targets in this region play critical roles in this activity. I also discovered that E1A requires a second patch of basic residues upstream of the canonical nuclear localization sequence (NLS) for nuclear localization. Thus, the previously described monopartite NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified. Finally, I also began investigating the global changes in gene expression mediated by the C-terminal targets of E1A during infection using next-generation RNAseq. These studies have expanded on our understanding of the mechanisms by which E1A reprograms the infected cell to induce oncogenic transformation