To infect target cells, HIV-1 must get past the gatekeeper of nuclear entry, the Nuclear Pore Complex (NPC). The human NPC is a 110 MDa complex, comprised of around 30 different nucleoporin proteins (Nups). One third of nucleoporins possess FG-motif repeats, regions of intrinsic disorder that are enriched for phenylalanine-glycine (‘FG’) dipeptides. These FG-Nups phase separate to form a selective barrier, which prevents the diffusion of molecules greater than 40 kDa.
HIV-1’s ability to infect non-dividing cells maps to the capsid, which has recently been shown to remain intact when crossing the nuclear envelope. Precisely how an intact capsid can subvert the selective barrier, despite being over 1000-times the size of the passive diffusion limit, is a critical knowledge gap. Transporters in the Karyopherin family can carry cargo larger than the 40 kDa cut-off through the NPC by specifically interacting with the FG repeats. Interestingly, the capsid is known to recruit cellular cofactors to a shared binding site by recognizing an exposed FG-motif. We hypothesised that the HIV capsid uses this binding site to directly engage with the FG motifs of a broad variety of Nups.
We have used a cell free expression system to produce ten human FG-repeat nucleoporins as GFP fusion proteins. Using our recently published fluorescence fluctuation spectroscopy screen we show that seven of the ten FG-NUPs interact directly with the HIV-1 capsid. These novel capsid cofactors can be competed off the capsid by the drugs PF74 and GS6207, which are known to target the FG-binding site. In addition, binding to a panel of known capsid mutations further indicates specificity for this pocket. Finally, using truncations of Nup proteins we show that weak, multivalent interactions occur throughout the FG domain suggesting a mechanism strikingly similar to that used by the karyopherin transporters.