Transcriptional transactivation turns human iPSC-derived macrophages into an adenovirus-producing cell state

Unlabelled: Macrophages conduct first-in-line defense against pathogens, including human adenovirus (AdV). AdVs cause respiratory disease, persist in immune cells, and, upon reactivation, are life-threatening to immunocompromised individuals. Here, our single-cell, single-virus experiments showed that AdV-type-C5 entry into human induced-pluripotent stem cell-derived macrophages is attenuated at cell binding and endosomal escape. A significant fraction (~30%) of the double-stranded viral DNA (vDNA) reaches the cell nucleus; however, it failed to efficiently express the immediate-early viral epigenetic regulator E1A. E1A transcription of silenced vDNA was rescued by E1A expression from a heterologous promoter of a superinfecting AdV, and allowed for full viral replication and progeny formation, even days post-infection, indicating long-lived infectivity of dormant vDNA. Bulk RNA-seq analyses showed that attenuated single AdV-C5 infections upregulated signaling, defense, and proinflammatory genes, whereas productive coinfections upregulated DNA replication and signaling pathways. Together, our data demonstrate that macrophages are a Trojan horse for AdV, notably independent of interferon, raising the possibility that macrophages function as a reservoir for AdV and reactivate dormant virus via epigenetic signals. Importance: AdV are widespread, cause severe respiratory disease, persist in immune cells, and, upon reactivation, cause life-threatening conditions in immunocompromised individuals. Here, we show that human macrophages are either protected or susceptible to AdV, depending on the cell state, notably in an interferon-independent manner. The decisive cell state switch is the viral immediate-early transcription modulator E1A, which turns a repressive state into a permissive one and allows for the transactivation of dormant AdV-C5 genomes and viral progeny production. The data raise the possibility that macrophages are a hub for AdV persistence and epigenetic reactivation , in line with the notion that these cells resist immune clearance and serve as reservoirs for HIV, herpesvirus, SARS-CoV-2, or rubella virus infections.