Adenovirus E4-ORF3-dependent relocalization of TIF1α and TIF1γ relies on access to the Coiled-Coil motif.

The adenovirus E4-ORF3 protein promotes viral replication by relocalizing cellular proteins into nuclear track structures, interfering with potential anti-viral activities. E4-ORF3 targets transcriptional intermediary factor 1 alpha (TIF1α), but not homologous TIF1β. Here, we introduce TIF1γ as a novel E4-ORF3-interacting partner. E4-ORF3 relocalizes endogenous TIF1γ in virus-infected cells in vivo and binds to TIF1γ in vitro. We used the homologous nature, yet differing binding capabilities, of these proteins to study how E4-ORF3 targets proteins for track localization. We mapped the ability of E4-ORF3 to interact with specific TIF1 subdomains, demonstrating that E4-ORF3 interacts with the Coiled-Coil domains of TIF1α, TIF1β, and TIF1γ, and that the C-terminal half of TIF1β interferes with this interaction. The results of E4-ORF3-directed TIF1 protein relocalization assays performed in vivo were verified using coimmunoprecipitation assays in vitro. These results suggest that E4-ORF3 targets proteins for relocalization through a loosely homologous sequence dependent on accessibility.