Antiviral defense and virus exclusion from the cell nucleus restrict foreign
nucleic acid influx and
infection. How the genomes of DNA viruses evade cytosolic pattern recognition and cross the nuclear envelope is incompletely understood. Here, we show that the virion
protein V of adenovirus functions as a linchpin between the genome and the capsid, thereby securing particle integrity. Absence of
protein V destabilizes cytoplasmic particles and promotes premature genome release, raising
cytokine levels through the
DNA sensor cGAS. Non-ubiquitinable V yields stable virions, genome misdelivery to the cytoplasm, and increased
cytokine levels. In contrast, normal
protein V is ubiquitinated at the nuclear pore complex, dissociates from the virion depending on the
E3 ubiquitin ligase Mib1 and the
proteasome, and allows genome delivery into the nucleus for
infection. Our data uncover previously unknown cellular and viral mechanisms of
viral DNA nuclear import in pathogenesis, vaccination, gene therapy, and synthetic biology.