Inhibition of mRNA nuclear export promotes SARS-CoV-2 pathogenesis

Significance This study shows the impact of inhibiting the mRNA nuclear export machinery on SARS-CoV-2 pathogenesis. By defining the mechanism and interaction surface between the SARS-CoV-2 Nsp1 protein and the mRNA export receptor NXF1, which mediates inhibition of cellular mRNA nuclear export, we were able to generate a mutant SARS-CoV-2 that does not inhibit mRNA nuclear export and is attenuated. Our results establish the importance of Nsp1-mediated mRNA export inhibition for SARS-CoV-2 replication and pathogenesis and reveal a surface on Nsp1 protein that can be potentially targeted for antiviral development.


Protein purification
GST-NXF1 117-619 -NXT1 wild-type and mutants were co-expressed in E. coli Rosetta cells (EMD Millipore, 70954).Protein expression was induced at an optical density at 600 nm of 0.6 with 0.5 mM IPTG at 18 °C and cells were incubated for 16 h.Cells were collected and lysed with a cell disruptor (Avestin) in a wash buffer (50 mM Tris, pH 8.0, 500 mM NaCl, 1 mM DTT) supplemented with a protease inhibitor mix containing 1 mM PMSF, 0.1 mM AEBSF, and 5 mg/L aprotinin.Cleared cell lysate was incubated with Glutathione Sepharose resins (Cytiva, 17075605), which were washed extensively with the wash buffer.Bound proteins were eluted from the Glutathione Sepharose resins with the wash buffer supplemented with 10 mM reduced glutathione, followed by digestion with GST tagged TEV protease (GST-TEV) at 4 °C.NXF1 117- 619 -NXT1 were applied to a HiTrap SP column (Cytiva) and eluted with a 150-400 mM NaCl gradient in a buffer containing 10 mM HEPES, pH 7.0, 1 mM DTT. HiTrap elution was passed through Glutathione Sepharose resins to remove undigested sample.NXF1 117-619 -NXT1 were further purified using a Superdex 200 column (Cytiva) equilibrated with 10 mM tris, pH 8.0, 300 mM NaCl, and 0.5 mM TCEP.GST-TEV digested NXF1 117-619 -NXT1 mutants were buffer exchanged to a buffer containing 50 mM Tris, pH 8.0, 500 mM NaCl, 1 mM DTT and passed through Glutathione Sepharose resins to remove GST and undigested sample.NXF1 117-619 -NXT1 mutants were further purified using a Superdex 200 column equilibrated with 10 mM tris, pH 8.0, 300 mM NaCl, and 0.5 mM TCEP.
Protein expression was induced at an optical density at 600 nm of 0.6 with 0.5 mM IPTG at 18 °C and cells were incubated for 16 h.GST-Nsp1 and GST-Nsp1 variants were first purified by glutathione-affinity chromatography, followed by a Superdex 200 column equilibrated with 10 mM tris, pH 8.0, 300 mM NaCl, and 0.5 mM TCEP.For the purification of untagged Nsp1 and Nsp1 D33K/E36K/E37K/E41K , glutathione-affinity purified GST-Nsp1 and GST-Nsp1 D33K/E36K/E37K/E41K were applied to a Source 15Q column (Cytiva) and eluted with a 75 mM to 450 mM NaCl gradient.
GST-Nsp1 pBpa45 was co-expressed with a tRNA synthetase/tRNA pair encoded by a plasmid pEVOL-pBpF (Addgene, 31190) in E. coli BL21 (DE3) (NEB, C2527).Arabinose was added to the cell culture to 0.02% (w/v) at an optical density at 600 nm of 0.3.Cells were spun down at an optical density at 600 nm of 0.6 and resuspended in 5-fold less volume media in the presence of 1 mM pBpa (AEchem) and 0.02% (w/v) arabinose.Cells were incubated at 16 °C for 1h and protein expression was then induced with 0.5 mM IPTG at 16 °C for 16h.Nsp1 pBpa45 was first purified by glutathione-affinity chromatography, followed by digestion with GST-TEV at 4 °C.
Sample was buffer exchanged to a buffer containing 50 mM Tris, pH 8.0, 500 mM NaCl, 1 mM DTT and passed through Glutathione Sepharose resins to remove GST and undigested sample.
Nsp1 pBpa45 was further purified using a Superdex 200 column equilibrated with 10 mM tris, pH 8.0, 300 mM NaCl, and 0.5 mM TCEP.All purified proteins were concentrated and stored at −80°C.

RNA fluorescence in situ hybridization (FISH)
For detection of poly(A) RNA cells, oligo-d(T) in situ hybridization was performed as we previously described (3).Images were acquired with a Zeiss Axiovert 200 M automated microscope using a 63X Plan-APOCHROMAT lens (1.4 numerical aperture) and captured by a Hamamatsu ORCAspark CMOS monochrome camera.Images were recorded sequentially for the antibody, RNA and DNA fluorescence channels respectively using a step size of 0.5 μm (total z size 10 μm).
Acquired z stack images were blind deconvolved using Autoquant X (Media Cybernetics).
For Sinai.After 24 hours of infection, cells were fixed according to the manufacturer's instructions.
The manufacturer's protocol for samples processed on coverslips in a 24-well plate was followed with additional modifications: A hybridization oven (HybEZ™ II Hybridization System, ACD) at 40 0 C was used for all steps requiring 40 0 C. A humidified staining tray (HybEZ™ II Hybridization System, ACD) was used for all steps requiring a humidified staining tray.Primary antibody was a mouse monoclonal anti-SARS-CoV1/2-N antibody (clone 1C7C7) and was used at a dilution of 1:300.Probes were used at a 1:100 dilution.Incubations were lengthened for steps 14 and beyond.
Step 18 was a 2.5 h incubation.
Step 26 was a 1.5 h incubation.
Step 29 was a 1.5 h incubation.
Step 31 was a 1.5 h incubation.Antifade Gold mounting media with DAPI was used for mounting.Glass slides from Matsunami (ref: SUMGP15) were used in these experiments.
Coverslips were sealed with clear nail polish after mounting.Samples were stored at 4 0 C in the dark before acquisition.Images were acquired the day after staining to avoid fading of the probe.
Images were acquired by confocal microscopy using the LSM-880 microscope.The image acquisition settings are 0.5 µm optical z-sections spanning a cell volume with a 63x oil objective.

Imaris-assisted image analysis for quantitative FISH
For quantification of poly(A) RNA, the Imaris software package Cells module (Bitplane, Version 9.8.2) was used for segmentation and signal analysis within the cytoplasm and nucleus.First, nuclei (Hoechst 33258) were segmented using an automated threshold (based on the intensity distribution histogram) in 405 nm laser line.Nuclei populations were filtered to remove large nuclei aggregates (upper nuclei volume threshold and lower threshold to manually remove fragments of nuclei at the edges of the stack) using the Imaris surface tool.Next, 3xFlag-tagged Nsp1 (Alexa 488 secondary) and total cellular poly(A) RNA signal (Cy5-poly(A) probe) were identified at 488 nm and 633 nm, respectively, using the Imaris surface tool.For objective cell body identification, high intensity and exposure thresholds were set for both 488 and 630 channels.The single cell distribution of poly(A) RNA signal was extracted and calculated as the intensity sum of voxels within the detected total cell volume or nuclear volume using the masking and statistic tools of Imaris software package Cells module.Brightness and contrast were linearly enhanced using Adobe Photoshop's Level tool.Statistical analysis was carried out using the one-way ANOVA with a Tukey post test (Prism 9, GraphPad).
For quantification of specific cellular mRNAs, the Imaris software package Cells module (Bitplane, Version 9.8.2) was used to identify and create a conventional 2-dimensional maximum intensity image of the nucleus (chromatin), SARS-CoV2 N and ViewRNA ISH signal.First, nuclei (DAPI) were segmented using an automated threshold (based on the intensity distribution histogram) in 405 nm laser line.Identified nuclei populations were filtered to remove large nuclei aggregates (upper nuclei volume threshold and lower threshold to manual remove fragments of nuclei at the edges of the stack) using the Imaris surface tool.Next, SARS-CoV2 N expression (protein marker for infected cells) and ViewRNA ISH signal for selected mRNA were identified in 488 nm laser line using Imaris surface tool and 467 nm laser line using the Imaris spot tool, respectively.Cell segmentation was performed in manual drawing mode at 1 mm vertex spacing.The seed spot size used was 0.2-0.7 mm.For objective mRNA foci center identification, automatic thresholds were used to filter raw spot quality.After identification of all mRNA spots in the cell, the nuclear mRNA spots were segmented, setting spot filter function to the "shortest distance to Nucleus", upper-threshold to 0, and turning the lower-threshold off.Brightness and contrast were linearly enhanced using Adobe Photoshop's Level tool.Statistical analysis was carried out using the one-way ANOVA with a Tukey post test (Prism 9, GraphPad).

Cell-based translation assay (including statistical analysis and Western blot conditions)
Fluc mRNA generation: to test the effect of Nsp1 on cellular translation we expressed FLAGtagged SARS-CoV-2 Nsp1 wildtype and indicated mutants in HEK293T cells and monitored translation of a co-transfected capped Firefly luciferase (Fluc) reporter mRNA.The Fluc mRNA was generated by in vitro transcription (IVT) using the mMESAGE mMACHINE T7 Transcription Kit (Invitrogen) and purified with the MEGAclear kit (Invitrogen) according to the manufacturer's protocol.The template for the IVT was generated by PCR from the pCAG-Luc plasmid (Addgene, #55764) using the indicated primer sequences: (T7-kozak-Fluc-Fw: 5'-TAATACGACTCACTATAGAGCCACCATGGAAGATGCCAAAAA-3'; PolyA-Fluc-Rv: 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTACACGGCGATCTTGCCG-3').
The size and quality of the transcript were analyzed by agarose gel electrophoresis.

Translation
Assay: HEK293T cells were cultured in 24-well plates and transfected with 500ng pClneo vector or pCl-neo SARS-CoV-2 Nsp1 wildtype or mutants.Plasmid DNA was transfected using TransIT-LT1 Transfection Reagent (Mirus) in Opti-MEM Reduced Serum Medium (Gibco).The firefly luciferase (Fluc) mRNA (250 ng) was transfected 24 hours after DNA transfection using Lipofectamine MessengerMAX (Invitrogen) in Opti-MEM Reduced Serum Medium (Gibco) according to the manufacturer's recommendations.Prior to mRNA transfection, cells were washed once with fresh Dulbecco's modified Eagle's medium (Corning) supplemented with 10% FBS (Peak Serum) and penicillin/streptomycin (Corning).At 10 hours after mRNA transfection, cells were lysed in Passive Lysis buffer (Promega) and the luminescence was quantified using Luciferase Assay System (Promega) according to the manufacturer's instructions.Fluc values were normalized to the values of the empty vector samples.To determine statistical significance, a one-way ANOVA with multiple comparisons using Dunnett's correction was performed.Nsp1 wildtype or Nsp1 mutant luciferase are compared to empty vector; p-value = 0.05.Western Blot: cell lysates from the translation assay were resuspended in 2X Laemmli sample buffer (Bio-Rad Laboratories) and boiled prior to loading.Samples were run on a 4-20% gradient polyacrylamide gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories) using the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories).Membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 detergent (TBS-t) with 5% nonfat dry milk.Primary antibodies were used at dilutions of 1:1000 in 3% bovine serum albumin in TBS-T.Metabolic labeling with Click-iT HPG Alexa 594, Fluorescent non-canonical amino acid tagging (FUNCAT) assayA549 cells were seeded at a density of 40,000/cells per well in a 24-well format on the top of round glass coverslips and transfected with 1µg pCI-Neo-3xFLAG or pCl-neo-3xFLAG SARS-CoV-2-Nsp1 wildtype or mutants using Lipofectamine 3000 reagent (Cat.no.18324012, Thermo Fisher

Table S2 . Analysis of Nsp1 pBPa45 crosslinks to NXF1 a
X=pBpa b Number in parenthesis for indicated site of crosslink gives spectral count that species.