SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection.

Many DNA viruses including polyomaviruses (PyVs) enter the host nucleus to cause infection, although how this is accomplished is unclear. To infect cells, the prototype PyV SV40 targets to the Nesprin-2 outer nuclear membrane protein and enters the nucleus via the nuclear pore complex (NPC). Host factors that function with Nesprin-2 to target SV40 to the nuclear membrane and drive NPC-dependent nuclear entry are unknown. Here we demonstrate that the SUN1 inner nuclear membrane protein acts coordinately with its binding-partner Nesprin-2 to target cytosol-localized SV40 to the nuclear membrane. Strikingly, despite localizing to the perinuclear space, the SUN domain of SUN1 plays a crucial role in Nesprin-2-dependent recruitment of cytosolic SV40. After targeting, SV40 binds to the NPC-associated importin receptor KPNA4, which translocates the virus into the nucleus. Our results reveal how a DNA virus exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the nucleus to cause infection. Author summary: Nuclear entry is required for most DNA viruses to cause infection, although the molecular mechanism of this step remains enigmatic. The DNA virus SV40 targets to the nuclear membrane by exploiting the Nesprin-2 outer nuclear membrane protein. In this study, we report that the SUN1 inner nuclear membrane protein functions with Nesprin-2 to target SV40 to the nuclear membrane, followed by nuclear entry of the virus via the action of the KNPA4 importin receptor.