The HIV capsid mimics karyopherin engagement of FG-nucleoporins

HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus. Remarkably, the intact HIV capsid is over one thousand times greater than the size-limit prescribed by the nuclear pore’s diffusion barrier. This barrier is a phase-separated condensate in the central channel of the nuclear pore and is comprised of intrinsically-disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides. Through multivalent FG-interactions, cellular karyopherins and their bound cargoes solubilise in this phase to drive nucleocytoplasmic transport. By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG-motifs from multiple nucleoporins and that this interaction licenses capsids to penetrate nucleoporin condensates. This karyopherin mimicry model resolves a key conceptual challenge for the role of the HIV capsid in nuclear entry, and explains how an exogenous entity much larger than any known cellular cargo can non-destructively breach the nuclear envelope.

Methods
Fluorescence Fluctuation Spectroscopy Data:

Data were collected as described here: https://dx.doi.org/10.1021/acs.analchem.0c04250?ref=pdf. Fluorescence traces were analyzed using custom software TRISTAN (Two Reagents Incident Spectroscopic Analysis, freely available on https://github.com/lilbutsa/Tristan)

Confocal Microscopy Imaging:   

Imaging was performed with Zeiss LSM 880 inverted laser scanning confocal microscope using a 63x oil immersion objective (NA=1.4) (Leica, Bensheim, Germany). The substrate-Nup98 reaction mixes were transferred into a 12 well silicone chamber (Ibidi) on 170 ± 5 µm cover slide. Z-stacks were taken around the centres of the phase separated Nup98 condensates (position with highest diameter) with sequential imaging at 488 and 568. Images were processed using ImageJ and Matlab.

Cryo-Electron Tomography

The grids were imaged on a Talos Arctica electron microscope (Thermo Fisher Scientific) operated at 200 kV acceleration voltage. Cryo-ET data were collected with single-axis tilt on a Falcon III direct electron detector (Thermo Fisher Scientific) in linear mode at a magnification of 28,000x with a pixel size of 5.23 Å.

Tilt series were collected using the dose-symmetric scheme8 from -60 to 60° at 3° intervals using Tomography software (Thermo Fisher Scientific) with the defocus value set at -10 µm. Total dose for each tilt series ranged from 60-70 e/Å2. Images of tilt series were binned twofold before tomograms were reconstructed. Three-dimensional reconstructions from tilt series were generated with the IMOD package9. Fiducial tracking was used to align the stack of tilted images.