Source data and statistical analysis.

Human adenoviruses (HAdVs) are widespread pathogens with the capacity to manipulate host cellular pathways, including critical tumor suppressor networks. During oncogenic cell transformation, the adenoviral E1B-55K protein serves as a multifunctional viral regulator that, inter alia, modulates both p53-dependent and -independent pathways – though this function has been disputed in the context of viral infection. Here, we elucidate the dual role of E1B-55K in disrupting host defenses, focusing on its impact on p53 signaling and interferon-stimulated genes (ISGs) during infection. Using RNA-seq and follow-up experimental validation in A549 (p53 wildtype) and H1299 (p53-null) cells infected with wildtype HAdV-C5 or an E1B-55K-deficient mutant, we show that E1B-55K suppresses p53-mediated transcriptional responses. Concurrently, E1B-55K modulates ISG expression in a context-dependent manner. Our results reveal that E1B-55K leverages cellular context to optimize viral replication by targeting a host tumor suppressor and indicate interference with innate immune pathways. Our study thereby uncovers a previously underappreciated aspect of E1B-55K function during infection, offering insights into its repressive activity and solidifying its role as a multifunctional viral oncoprotein with broader implications for the HAdV replication cycle.