Nuclear entry represents the final and decisive infection step for most DNA viruses, although how this is accomplished by some viruses is unclear. Polyomavirus SV40 transports from the cell surface through the endosome, the endoplasmic reticulum, and the cytosol from where it enters the nucleus to cause infection. Here we elucidate the nuclear entry mechanism of SV40. Our results show that cytosol-localized SV40 is targeted to the nuclear envelope by directly engaging Nesprin-2 of the linker of nucleoskeleton and cytoskeleton (LINC) nuclear membrane complex. Additionally, we identify the NUP188 subunit of the nuclear pore complex (NPC) as a new Nesprin-2-interacting partner. This physical proximity positions the NPC to capture SV40 upon release from Nesprin-2, enabling the channel to facilitate nuclear translocation of the virus. Strikingly, SV40 disassembles during nuclear entry, generating a viral genome-VP1-VP3 subcomplex that efficiently crosses the NPC to enter the nucleus. Our results reveal how two major nuclear membrane protein complexes are exploited to promote targeting and translocation of a virus into the nucleus. Author summary: Although many DNA viruses cause infection by moving from the cell surface to the nucleus of the host cell, the molecular basis of this process remains enigmatic in many instances. Polyomavirus is a DNA tumor virus that is responsible for several debilitating human diseases, but how it reaches the nucleus to cause infection is mysterious. Here we discover that two major nuclear membrane protein complexes–LINC and NPC–act cooperatively to drive nuclear entry of SV40, the prototype polyomavirus. Illuminating a critical polyomavirus entry step may lead to novel strategies to combat polyomavirus-induced diseases. [ABSTRACT FROM AUTHOR]