Structural basis for Sarbecovirus ORF6 mediated blockage of nucleocytoplasmic transport

Figures

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  Fig. 1. Peptides containing the C-terminal tails of SARS-CoV-2 and SARS-CoV ORF6 proteins interact with the Rae1-Nup98 complex at nanomolar affinity. a Diagrams of domain organization of Rae1, Nup98 and Sarbecovirus ORF6 proteins with annotations. b Final purification step of Rae1 WD Repeat—Nup98GLEBS complex. Size-exclusion chromatography profile of Rae1-Nup98 complex. c SDS-PAGE analysis of the eluate from the size-exclusion chromatography shown in b. Results shown are representative of three independent experiments. d Sequence of SARS-CoV-2 and SARS-CoV ORF6 CTT peptides (C1-C4); key methionine M58 is highlighted in red; two C3 peptides that co-crystalized with Rae1-Nup98 complex are highlighted with yellow background. e–h ITC titrations between SARS-CoV-2 ORF6 C1-C4 peptides and Rae1-Nup98. i ITC experiment titration between SARS-CoV ORF6 C3 peptide and Rae1-Nup98. Source data are provided as a Source data file.

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  Fig. 2. Crystal structures of SARS-CoV-2 and SARS-CoV ORF6 CTT peptides complexed with Rae1-Nup98. a Left, ribbon model of the SARS-2 ORF6 C3 peptide complexed with Rae1-Nup98. Right, magnified view of the peptide-protein interaction. The Rae1 (blue)—Nup98 (green) complex is shown with molecular surface, the bound SARS-CoV-2 ORF6 C3 is shown with magenta stick model; a composite omit map associated with the peptide is shown with black meshes. b Left, ribbon model of the SARS-CoV ORF6 C3 peptide complexed with Rae1-Nup98. Right, magnified view of the peptide-protein interaction. The Rae1 (blue)—Nup98 (green) complex is shown with molecular surface, and the bound SARS-CoV ORF6 C3 is shown with orange stick model; a composite omit map associated with the peptide is shown with black meshes. c Detailed interactions between Rae1 (blue stick model) and the SARS-CoV-2 ORF6 C3 peptide (magenta stick model). Residues involved in the interaction are labeled, and intermolecular hydrogen bonds are shown with the dashed lines. d Detailed interactions between Rae1 (blue stick model) and the SARS-CoV ORF6 C3 peptide (orange stick model). Residues involved in the interaction are labeled, and…

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  Fig. 3. Investigation of the ORF6-derived peptides binding to the Rae1-Nup98 complex and their inhibition of RNA binding with Rae1-Nup98. a Histogram showing binding affinity of a selection of peptides derived from the CTT or NTE of different proteins to the Rae1-Nup98 complex. Key residues are highlighted in red and green. While the y axis indicates names and sequence of different peptides, the x-axis indicates the folds (folds of binding affinity) as compared with the affinity of SARS-CoV-2 ORF6 C3 peptide (Kd = 0.24 μM). Thus, the folds of binding affinity were calculated: 0.24 μM / Kd of the indicated peptide. b–j ITC experiments showing the interaction between each peptide and the Rae1-Nup98 complex. The binding affinity measured from each experiment is indicated. k, l EMSAs showing the ability of Sarbecovirus ORF6 CTTs (C3 peptide) to disrupt ssRNA binding with Rae1-Nup98. After pre-incubating the FAM labeled 10-mer poly(U) ssRNA with Rae1-Nup98 complex, increasing amounts (0–64 μM) of SARS-CoV-2 or SARS-CoV ORF6 C3 peptides were added to the mixtures to allow competition with the ssRNA. The resulting mixtures were analyzed by native-PAGE and visualized using Typhoon scanner. This…

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  Fig. 4. Interactions with Rae1-Nup98 are important for ORF6-mediated disruption of nucleocytoplasmic transport. a HEK-293T cells were co-transfected with the indicated plasmids, and 48 h later, Co-IP analysis was performed to examine the interaction between Rae1-Nup98 and wild-type (WT) or mutant viral proteins. Results shown are representative of three independent experiments. b HEK-293T cells were transfected with pEGFP-C1 and individual plasmid expressing WT or mutant ORF6, ORF10, or M. GFP expression were analyzed by fluorescence microscopy. All fluorescence images at 40 times magnification (40×). Results shown are representative of three independent experiments. c Quantification of the fluorescence intensity in b with Image J. Data are representative of three independent experiments and shown as the mean ± SD. (*p < 0.05, ***p < 0.001, ****p < 0.0001; two tailed Student’s t-test, n = 3). d The cytoplasm and nuclear RNAs were extracted from transfected cells in b, and the levels of GFP transcripts were quantified by RT-PCR. The Nu/Cyto ratio is plotted as the mean ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; two tailed Student’s t-test, n = 3). e, f The effects of ORF6…