The infection of Mib1-KO cells with AdV-C5-V-KR was more efficient than wild-type AdV-C5, but not as efficient as AdV-C5-∆V, suggesting that Mib1 is involved in ubiquitination of an additional target besides protein V to gate nuclear import of the vDNA from capsids containing protein V. Together, the results show that the vDNA core–associated protein V serves as a linchpin securing the stability of cytoplasmic AdV and becomes a Mib1-dependent ubiquitin-responsive target at the NPC facilitating the nuclear import of vDNA upon virion rupture. The gene discussed is C5; the disease is infection.