Exploring the Effects of Adenovirus E4-ORF3 on Protein Localization and Cellular Gene Expression

The Adenovirus E4-ORF3 protein facilitates virus replication through the relocalization of cellular proteins into elongated, nuclear structures termed tracks. E4-ORF3 targets proteins such as Mre11-Rad50-Nbs1 (MRN) complex members, PML, TIF1α, and TIF1γ and is thought to inhibit associated antiviral properties. To further characterize the role that E4-ORF3 plays in facilitating Ad replication, this dissertation focuses on how E4-ORF3 selects cellular proteins for track localization and what effects these rearrangement events have on gene expression. E4-ORF3 selectively targets TIF1α and TIF1γ; but not related TIF1β. The structural similarity of these proteins yet differential ability to interact with E4-ORF3 make them an attractive vehicle for studying how E4-ORF3 selects proteins for track localization. Generating a series of truncated EYFP-fusion constructs and protein chimeras, I determined that E4-ORF3 targets the coiled-coil of all three tested TIF1 proteins. The C-terminal portion of TIF1β, consisting of the middle region and PHD-Bromo domain, interferes with the ability of E4-ORF3 to access the coiled-coil motif. These results suggest that E4-ORF3 targets a loosely homologous sequence in a manner dependent on protein context.;E4-ORF3 relocalizes a number of cellular proteins associated with transcriptional regulatory activities. An attractive hypothesis suggests that through interaction with these proteins, E4-ORF3 alters their ability to regulate gene expression in a manner that promotes viral replication. I performed a series of microarray time course experiments to study the effect of E4-ORF3 on cellular gene expression yielding a list of 409 genes that display E4-ORF3-dependent expression changes. Enrichment analysis of genes differentially expressed at 24 hours post-induction relative to 6 hours post-induction suggests E4-ORF3 influences factors involved in signal transduction and cellular defense. The introduction of mutant versions of E4-ORF3 reveals that track formation, but not the sequestration of the MRN complex, is necessary to induce these expression changes. By generating knockdown cells, we demonstrate that E4-ORF3 may relieve the PML-dependent inhibition of RGS2 gene expression.