1.
Quantification of nuclear transport inhibition by SARS-CoV-2 ORF6 using a broadly applicable live-cell dose-response pipeline.
Abstract:
SARS coronavirus ORF6 inhibits the classical nuclear import pathway to antagonize host antiviral responses. Several models were proposed to explain its inhibitory function, but quantitative measurement is needed for model evaluation and refinement. We report a broadly applicable live-cell method for calibrated dose-response characterization of the nuclear transport alteration by a protein of interest. Using this method, we found that SARS-CoV-2 ORF6 is ∼5 times more potent than SARS-CoV-1 ORF6 in inhibiting bidirectional nuclear transport, due to differences in the NUP98-binding C-terminal region that is required for the inhibition. The N-terminal region was also required, but its membrane binding function was dispensable, since loss of the inhibitory function due to N-terminal truncation was rescued by forced oligomerization using a soluble construct. Based on these data, we propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the FG domains of NUP98 at the nuclear pore complex.
2.
Livecell reporters reveal bidirectional acceleration of nucleocytoplasmic transport by O-GlcNAc modification of the nuclear pore complex.
Abstract:
Macromolecular transportacross the nuclear envelope is fundamental to eukaryotic cells and depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains apermeability barriermade of phenylalanine-glycine (FG) repeat domainsthat selectively facilitatesthe permeation ofcargoes bound to nuclear transport receptors (NTRs)1,2.The NPC is enriched in O-linked N-acetylglucosamine (O-GlcNAc) modification3-8, but itsfunctional rolein nucleocytoplasmic transport isunclear. We developed high-throughput assaysbased on optogenetic probes to quantify the kinetics of nuclear import and exportin living human cells and showedthat the O-GlcNAc modification of the NPC accelerates the nucleocytoplasmic transport in both directions.Super-resolution imaging of O-GlcNAc revealed strong enrichmentat the FG barrier ofthe NPC channel. O-GlcNAcmodificationalso promoted the passive permeation of a small,inert protein through NPCs.Our results suggest that O-GlcNAc modification acceleratesnucleocytoplasmic transport by enhancingthe non-specific permeabilitythe FG-repeat barrier.
