The human nuclear pore complex (NPC) is a ~110 MDa protein complex comprised of 32 different nucleoporins (Nups). The NPC mediates selective passage of components between the cytoplasm and the nucleus by providing a passive diffusion-barrier for molecules bigger than 40 kDa. This selectivity is maintained by a group of intrinsically disordered phenylalanine-glycine (FG)-rich FG-Nups, which undergo liquid-liquid phase separation to create the NPC’s diffusion barrier. Proteins of the karyopherin family mediate active transport of larger macromolecules by specifically interacting with FG dipeptides in the FG-rich selectivity barrier.
Astonishingly, the 35 MDa Capsid (CA) of HIV-1 has been found to cross the nuclear envelope via translocation through the NPC. We propose that the CA can access the FG-rich selectivity barrier by adapting a karyopherin like mechanism.
We focused on in-vitro assays using recombinant CA and the FG-domain of Nup98, a key player in the NPC due to its high abundance and importance in maintaining the NPC’s structural integrity. Consistent with previous reports, we show that at mM concentrations Nup98 spontaneously phase separates into µm sized spherical condensates. Importantly, these condensates maintain the selective properties found in the NPC by excluding proteins bigger than the 40 kDa size limit but allowing specific interaction of the karyopherin importin-b.
Using confocal microscopy and the Nup98 model system, we found that intact CA lattices bind and partition into FG condensates within minutes of incubation. We furthermore demonstrated that addition of the well characterised drugs PF74 and GS6207 completely abolished binding and diffusion of CA into the Nup98 condensates, indicating that migration is due to an FG-specific binding pocket on the CA lattice surface.
These results suggest that the HIV CA specifically interacts and diffuses through the NPC selectivity barrier with a mechanism like that of the karyopherins.