A DNA-origami nuclear pore mimic reveals nuclear entry mechanisms of HIV-1 capsid

Abstract

The capsid of human immunodeficiency virus 1 (HIV-1) plays a pivotal role in viral nuclear import, but the mechanism by which the viral core passages the nuclear pore complex (NPC) is poorly understood. Here, we use DNA-origami mimics of the NPC, termed NuPODs (NucleoPorins Organized by DNA), to reveal the mechanistic underpinnings of HIV-1 capsid nuclear entry. We found that trimeric interface formed via three capsid protein hexamers is targeted by a triple-arginine (RRR) motif but not the canonical phenylalanine-glycine (FG) motif of NUP153. As NUP153 is located on the nuclear face of the NPC, this result implies that the assembled capsid must cross the NPC in vivo. This hypothesis is corroborated by our observations of tubular capsid assemblies penetrating through NUP153 NuPODs. NUP153 prefers to bind highly curved capsid assemblies including those found at the tips of viral cores, thereby facilitating capsid insertion into the NPC. Furthermore, a balance of capsid stabilization by NUP153 and deformation by CPSF6, along with other cellular factors, may allow for the intact capsid to pass NPCs of various sizes. The NuPOD system serves as a unique tool for unraveling the previously elusive mechanisms of nuclear import of HIV-1 and other viruses.

Nuclear Import of HIV-1

“…This binding mode suggests that, at least, a partially assembled capsid, beyond the CA hexamer, is required for stable NUP153-capsid interaction. Moreover, the presence of NUP153 appeared to stabilize the assembled CA lattice [57], which further suggests that NUP153 protects the integrity of the CA capsid during the capsid passage through the NPC. Given the location of NUP153 on the nuclear side of the NPC and the necessity of at least partially intact capsids for interaction, NUP153 may stabilize the capsid at the late stage of nuclear import.…”

“…Structural studies have shown that this NUP153 FG-motif is engaged at the NTD-CTD interface between two CA subunits of the hexamer, albeit with a relatively weak binding affinity (Kd) of ~50 µM [60,63]. It was recently identified that the C-terminal 65 residues of NUP153 interacted with CA assemblies beyond hexamers at a much higher affinity [57]. Notably, this strong interaction is achieved through a bipartite motif of NUP153, containing both the canonical FG-motif and an additional triple-arginine (RRR) motif [57].…”

“…It was recently identified that the C-terminal 65 residues of NUP153 interacted with CA assemblies beyond hexamers at a much higher affinity [57]. Notably, this strong interaction is achieved through a bipartite motif of NUP153, containing both the canonical FG-motif and an additional triple-arginine (RRR) motif [57]. Interestingly, the canonical FG-motif in NUP153 contributes modestly to the capsid interaction, and the other FG-repeats in NUP153 do not enhance binding.…”

A DNA-origami NanoTrap for studying the diffusion barriers formed by Phe-Gly-rich nucleoporins

“…6,41 Our recent work also showed that the tip of the megadalton-sized HIV capsid can at least insert into the NPC-mimicking NuPOD. 42 The permeability properties of Nsp1 that we measured in the NanoTraps seem to be more in line with a soft-diffusion barrier type mechanism -one with a continuum of penetration rates that negatively correlates with increasing molecular weights of entering molecules. These observations are consistent with entropic barrier models where the dynamic nature of unstructured and flexible FG-nup filaments effectively occlude the channel.…”

“…6, 41 Our recent work also showed that the tip of the megadalton-sized HIV capsid can at least insert into the NPC-mimicking NuPOD. 42…”