Methods Details

Vero E6 cell infection for proteomic analysis
Vero E6 cells were seeded using 2x106 cells in T25 flasks. The following day cells were either mock infected or infected with SARS-CoV-2 at a MOI of 1 in serum-free DMEM at 37°C for 1 hour. After absorption the 0 hour samples were lysed immediately, while the media for other samples was replaced with 2% FBS / DMEM (Invitrogen) and incubated at 37°C for times indicated before lysis.

Cell lysis and digestion
Cells were lysed using 1% IGEPAL (Sigma) in PBS (Invitrogen) for 20 minutes at room temperature (RT) to inactivate the virus. These specific lysis conditions were used as this was the approved virus inactivation protocol. Proteins contained in the cell lysate were then immediately precipitated using 90% methanol (v/v) (Sigma) by centrifugation at 20,000x g for 10 minutes. The protein pellets were frozen at −80°C. Precipitated proteins were resuspended in lysis buffer (8 M urea, 100 mM ammonium bicarbonate (ABC), 150 mM NaCl, protease inhibitor (mini-cOmplete, Roche) and phosphatase inhibitors (phosSTOP, Roche). Tris-(2-carboxyethyl)phosphine (TCEP) was added to a final concentration of 4 mM. DNA was sheared via probe sonication, on ice, at 20% amplitude for 20 s., followed by 10 s of rest. This process was performed a total of three times. Following sonication, protein concentration was determined using Bradford assay. Iodoacetamide (IAA) was added to each sample to a final concentration of 10 mM, and samples were incubated in the dark at room temperature (RT) for 30 minutes. Excess IAA was quenched by the addition of dithiothreitol (DTT) to 10 mM, followed by incubation in the dark at RT for 30 minutes. Samples were then diluted with 0.1 M ABC (pH = 8.0) to a final urea concentration of 2 M. Trypsin (Promega) was added at a 1:100 (enzyme:protein w:w) ratio and digested overnight at 37°C with rotation. Following digestion, 10% trifluoroacetic acid (TFA) was added to each sample to a final pH ∼2. Samples were desalted under vacuum using Sep Pak tC18 cartridges (Waters). Each cartridge was activated with 1 mL 80% acetonitrile (ACN)/0.1% TFA, then equilibrated with 3 × 1 mL of 0.1% TFA. Following sample loading, cartridges were washed with 4 × 1 mL of 0.1% TFA, and samples were eluted with 4 × 0.5 mL 50% ACN/0.25% formic acid (FA). 20 μg of each sample was kept for protein abundance measurements, and the remainder was used for phosphopeptide enrichment. Samples were dried by vacuum centrifugation.

Phosphopeptide enrichment
For each sample batch, 400 μL (30 μL per sample) of 50% Superflow bead slurry (QIAGEN) was added to a 2 mL bio-spin column. Beads were incubated with 4 × 500 μL of 100 mM EDTA for 30 s, washed with 2 × 500 μL H2O, incubated 4 × 500 μL with 15 mM FeCl3 for 1 minute, washed 3 × 500 μL H2O, and washed once with 500 μL of 0.5% FA to remove residual Fe. Beads were resuspended in 600 μL of H2O and 60 μL were aliquoted into a C18 NEST column that had been equilibrated with 150 μL of 80% ACN, 0.1% TFA. 1 mg of digested peptides were resuspended in 75% ACN/0.15% TFA and incubated with beads for 2 minutes, mixed by pipetting and incubated again for 2 minutes. Beads were washed 4 × 200 μL with 80% ACN, 0.1% TFA, then washed 3 × 200 uL with 0.5% FA, incubated 2 × 200 μL with 500 mM potassium phosphate buffer pH 7 and incubated 2 × 200 μL with 0.5% FA for 15 seconds. Phosphopeptides were eluted by centrifugation at 3000 RPM for 30 seconds with 2 × 75 uL of 50% ACN, 0.1% FA.

Mass spectrometry data acquisition
Digested samples were analyzed on an Orbitrap Exploris 480 mass spectrometry system (Thermo Fisher Scientific) equipped with an Easy nLC 1200 ultra-high pressure liquid chromatography system (Thermo Fisher Scientific) interfaced via a Nanospray Flex nanoelectrospray source. For all analyses, samples were injected on a C18 reverse phase column (25 cm x 75 μm packed with ReprosilPur 1.9 μm particles). Mobile phase A consisted of 0.1% FA, and mobile phase B consisted of 0.1% FA/80% ACN. Peptides were separated by an organic gradient from 5% to 30% mobile phase B over 112 minutes followed by an increase to 58% B over 12 minutes, then held at 90% B for 16 minutes at a flow rate of 350 nL/minute. Analytical columns were equilibrated with 6 μL of mobile phase A. To build a spectral library, one sample from each set of biological replicates was acquired in a data dependent manner. Data dependent analysis (DDA) was performed by acquiring a full scan over a m/z range of 400-1000 in the Orbitrap at 60,000 resolving power (@200 m/z) with a normalized AGC target of 300%, an RF lens setting of 40%, and a maximum ion injection time of 60 ms. Dynamic exclusion was set to 60 seconds, with a 10 ppm exclusion width setting. Peptides with charge states 2-6 were selected for MS/MS interrogation using higher energy collisional dissociation (HCD), with 20 MS/MS scans per cycle. For phosphopeptide enriched samples, MS/MS scans were analyzed in the Orbitrap using isolation width of 1.3 m/z, normalized HCD collision energy of 30%, normalized AGC of 200% at a resolving power of 30,000 with a 54 ms maximum ion injection time. Similar settings were used for data dependent analysis of samples used to determine protein abundance, with an MS/MS resolving power of 15,000 and a 22 ms maximum ion injection time. Data-independent analysis (DIA) was performed on all samples. An MS scan at 60,000 resolving power over a scan range of 390-1010 m/z, a normalized AGC target of 300%, an RF lens setting of 40%, and a maximum injection time of 60 ms was acquired, followed by DIA scans using 8 m/z isolation windows over 400-1000 m/z at a normalized HCD collision energy of 27%. Loop control was set to All. For phosphopeptide enriched samples, data were collected using a resolving power of 30,000 and a maximum ion injection time of 54 ms. Protein abundance samples were collected using a resolving power of 15,000 and a maximum ion injection time of 22 ms.

Spectral library generation and raw data processing
Raw mass spectrometry data from each DDA dataset were used to build separate libraries for DIA searches using the Pulsar search engine integrated into Spectronaut version 13.12.200217.43655 (Bruderer et al., 2015) by searching against a database of Uniprot Chlorocebus sequences (19,136 proteins, downloaded April 3, 2020) and 29 SARS-CoV-2 protein sequences translated from genomic sequence downloaded from GISAID (accession EPI_ISL_406596, downloaded April 7, 2020) with two mutations (G22661T Spike V367F and G26144T ORF3a G251V) detected by RNASeq analysis of virus stocks. For protein abundance samples, data were searched using the default BGS settings, variable modification of methionine oxidation, static modification of carbamidomethyl cysteine, and filtering to a final 1% false discovery rate (FDR) at the peptide, peptide spectrum match (PSM), and protein level (Elias and Gygi 2007). For phosphopeptide enriched samples, BGS settings were modified to include phosphorylation of S, T, and Y as a variable modification. The generated search libraries were used to search the DIA data. For protein abundance samples, default BGS settings were used, with no data normalization performed. For phosphopeptide enriched samples, the Significant PTM default settings were used, with no data normalization performed, and the DIA-specific PTM site localization score in Spectronaut was applied.

Immunofluorescence microscopy
Caco-2 cells seeded on glass coverslips were infected with SARS-CoV-2 Isolate Muc-IMB-1/2020, second passage on Vero E6 cells (2x106 PFU/mL) at an MOI of 0.1 or 0.01. At 24 hours post-infection cells were washed with PBS and fixed in 4% paraformaldehyde in PBS for 20 minutes at RT, followed by permeabilization with 0.3% Triton X-100 in PBS for 10 minutes at RT and blocking in 5% fetal calf serum in PBS for 1 hour at RT. Incubation with primary antibodies against CK2α (Abcam, ab70774, 1:500), SARS-CoV membrane (M) protein (Rockland, #100-401-A55, 1:500) and SARS-CoV nucleocapsid (N) protein (Rockland, #200-401-A50, 1:1000) was performed for 1 hour at RT. After washing with PBS, cells were incubated with AF568-labeled goat-anti-rabbit (Invitrogen, #A11011) and AF647-labeled goat-anti-mouse (Invitrogen, #A21235) secondary antibodies (1:200) as well as AF488-labeled Phalloidin (Hypermol, #8813-01, 1:250) for 2 hours at RT. Fluorescence images were generated using a LSM800 confocal laser-scanning microscope (Zeiss) equipped with a 63X, 1.4 NA oil objective and Airyscan detector and the Zen blue software (Zeiss) and processed with Zen blue software and ImageJ/Fiji. For 3D-reconstruction, cells were fixed and stained as indicated and imaged as z stack with 0.15 μm sections. Z stack was processed using Imaris software 64x 9.5.1 and displayed as MIP. To quantify colocalization between casein kinase II and N protein colocalization events in filopodia of SARS-CoV-2 infected Caco-2 cells were counted. 42 ± 19 % of the N protein particles detected in filopodia colocalize with CK2. Length of filopodia was measured using Metamorph (Version 7.8). Distance was measured starting at cortical actin to the tip of Filopodia.

Electron microscopy
Vero E6 cells were seeded overnight and then infected for 24 hours with SARS-CoV-2 isolate nCoV-WA1-2020 on silicon chips for scanning electron microscopy or Thermanox for transmission electron microscopy. For scanning electron microscopy (SEM), cells were fixed with Karnovsky’s formulation of 2% paraformaldehyde/2.5% glutaraldehyde in 0.1 M Sorenson’s phosphate buffer, and then post-fixed with 1.0% osmium tetroxide/0.8% potassium ferrocyanide in 0.1 M sodium cacodylate buffer washed with 0.1 M sodium cacodylate buffer then stained with 1% tannic acid in dH2O. After additional buffer washes, the samples were further osmicated with 2% osmium tetroxide in 0.1M sodium cacodylate, then washed with dH2O. Specimens were dehydrated with a graded ethanol series from 50%, 75%, 100% x 3 for 5 minutes each, critical point dried under CO2 in a Bal-Tec model CPD 030 Drier (Balzers, Liechtenstein), mounted with double sided carbon tape on aluminum specimen mounts (Ted Pella), and sputter coated with 35 Å of iridium in a Quorum EMS300T D sputter coater (Electron Microscopy Sciences, Hatfield, PA) prior to viewing at 5 kV in a Hitachi SU-8000 field emission scanning electron microscope (Hitachi, Tokyo, Japan). For transmission electron microscopy (TEM), specimens were fixed as described above for scanning electron microscopy and additionally stained overnight with 1% uranyl acetate at 4°C after the second osmium staining and then dehydrated with the same graded ethanol series, and embedded in Spurr’s resin. Thin sections were cut with a Leica UC7 ultramicrotome (Buffalo Grove, IL) prior to viewing at 120 kV on a FEI BT Tecnai transmission electron microscope (Thermofisher/FEI, Hillsboro, OR). Digital images were acquired with a Gatan Rio camera (Gatan, Pleasanton, CA).

N overexpression in Vero E6 cells
The N-protein cassette was subcloned from pLVX-EF1a-SARS-CoV-2-N-2xStrep-IRES-Puro (Gordon et al., 2020) into pLVX-TetOne-Puro (Takara) using the restriction enzymes EcoRI and BamHI to create pLVX-TetOne-Puro-SARS-CoV-2-N-2xStrep. Sequence integrity was confirmed by Sanger sequencing (GeneWiz). To produce lentiviruses, HEK293T cells (50% confluent in a T175 flask) were transfected with 5 μg of each of the pLVX-TetOne-Puro lentiviral plasmids, 3.33 μg Gag-Pol packaging construct, and 1.66 μg VSV-G envelope (pMD2.G, Addgene) using PolyJet (SignaGen). Culture supernatant was precipitated with a final concentration of 8.5% PEG-6000 and 0.3M sodium chloride (NaCl), incubated at 4°C for 2-4 hours. Virions were pelleted at 3500 rpm for 20 minutes in a spin bucket rotor, suspended in 0.5 mL 1xPBS, and aliquoted for storage at 80°C. Vero E6 cells were seeded in T75 flasks at 50% confluence and transduced with 200 μL precipitated lentivirus derived from pLVX-TetOne-Puro-SARS-CoV-2-N-2xStrep or pLVX-TetOne-Puro empty vector. 48 hours post transduction, 10 μg/mL Puromycin was added to cultures to select transduced cells. Polyclonal stable cell lines were seeded into 15 cm dishes in triplicate and treated with 1 μg/mL doxycycline for 48 hours. Cells were washed in ice cold PBS and harvested by scraping in cold PBS. Cell pellets were lysed directly (8 M urea, 100 mM ammonium bicarbonate (ABC), 150 mM NaCl, protease inhibitor (mini-cOmplete, Roche) and phosphatase inhibitors (phosSTOP, Roche), and protein digestion and phosphopeptide enrichment was performed as described above for SARS-CoV-2 infected Vero E6 cells. During the analysis, one of three replicates of the N-overexpressed samples was found to be an outlier based on principal component analysis (as in Figures S1A and S1B) and was removed. N-overexpression (n = 2) was compared to empty vector (EV) controls (n = 3) during analysis (full data available in Table S1). Kinase activity predictions were also performed the same as for viral infection phosphoproteomics (full data available in Table S4).

Cell cycle analysis
1x105 Vero E6 cells were seeded per sample. The following day cells were either mock infected or infected with SARS-CoV-2 (isolate BetaCoV/France/IDF0372/2020) at an MOI of 1. The samples were incubated for 24 hours before detaching cells using Trypsin 0.05% (Thermo). Cells were pelleted at 500x g for 2 minutes, followed by fixation using 4% Formalin (Thermo) for 30 minutes. Cells were washed 2x with PBS (Thermo) before permeabilizing using 0.1% Triton X-100 (Thermo) for 20 minutes. Finally, cells were pelleted again and incubated with 1 mg/mL 4′,6-diamidino-2-phenylindole (DAPI; Thermo) in PBS for 30 minutes before analyzing cell cycle using fluorescence by flow cytometry using the BD FACSymphony with a violet laser (405 nm) and 450/50 nm filter. Gating was performed by first selecting singlets by gating highly correlated cells in an SSC-A versus SSC-H plot. Next, typical cellular morphology was gated using a FSC-A versus SSC-A plot. These singles were then used to draw histograms which were gated for G0/G1, S, and G2/M phases.

SARS-CoV-2 infections in ACE2-A549 cells and lysis for cytokine analysis
Approximately 5 × 105 A549-ACE2 cells were pre-treated with either DMSO or SB203580 (0.01, 0.1, 1, 10 μM final concentration). After 1 hour of pre-treatment, cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at an MOI of 0.1 while maintaining inhibitor concentrations in the media for 30 hours post infection. The supernatants from infected cells were analyzed by multiplexed ELISA for the presence of secreted cytokines and chemokines. For RNA analysis, cells were lysed in TRIzol and total RNA was extracted and DNase treated using the Direct-zol RNA Miniprep Plus kit (Zymo Research) according to the manufacturer’s instructions. For protein analysis, whole cell lysates were obtained by lysis in RIPA buffer and analyzed by SDS-PAGE and western blot.

Cytokine RT-qPCR analysis
Gene expression of selected cytokines as well as SARS-CoV-2 replication was quantified by RT-qPCR. Reverse transcription of extracted total RNA samples was performed using oligo (dT) primers and SuperScript IV Reverse Transcriptase according to the manufacturer’s instructions. Quantitative real-time PCR analysis of cDNA samples was performed using KAPA SYBR FAST qPCR Master Mix (2X) Universal on a LightCycler 480 Instrument II (Roche). For viral quantification, primers specifically targeting the subgenomic viral N RNA were used. ΔCT values were determined relative to the ACTB and ΔΔCT values were normalized to the average of mock infected samples (for host genes) or to infected DMSO treated samples (for viral replication). Error bars indicate the standard deviation of the mean from three independent biological replicates.

Multiplexed cytokine ELISA
Supernatants from infected cells were evaluated for 34 cytokines/chemokines by multiplex ELISA for the following analytes: CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL7/MCP-3, CCL8/MCP-2, CCL11/Eotaxin, CCL13/MCP-4, CCL20/MIP-3α, CXCL1/GROα, CXCL2/GROβ, CXCL5/ENA-78, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CXCL16, IL-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-7, IL-10, IL-12p70, IL-15, IL-16, IL-17A, IL-22, GM-CSF, MMP-9, S100A8, TNFα, TGFβ, and Trappin-2/Elafin. All antibodies and cytokine standards were purchased as antibody pairs from R&D Systems (Minneapolis, MN) or Peprotech (Rocky Hill, NJ). Individual magnetic Luminex bead sets (Luminex Corp, CA) were coupled to cytokine-specific capture antibodies according to the manufacturer’s recommendations. Samples were analyzed on a Luminex MAGPIX platform and quantified using a standard curve. For each bead region, > 50 beads were collected per analyte. The median fluorescence intensity of these beads was recorded for each bead and was used for analysis using a custom R script and a 5P regression algorithm.

SARS-CoV-2 viral quantification via antibody staining in presence of inhibitors
(Mount Sinai, New York). Two thousand (2,000) Vero E6 cells were seeded into 96-well plates and incubated for 24 hours. Two hours before infection, the medium was replaced with 100 μL of DMEM (2% FBS) containing the compound of interest at concentrations 50% greater than those indicated, including a DMSO control. Plates were then transferred into the BSL-3 facility and 100 PFU of SARS-CoV-2 (MOI 0.025) was added in 50 μL of DMEM (2% FBS), bringing the final compound concentration to those indicated. Plates were then incubated for 48 hours at 37°C. After infection, supernatants were removed and cells were fixed with 4% formaldehyde for 24 hours prior to being removed from the BSL-3 facility. The cells were then immunostained for the viral NP protein (anti-sera produced in the Garcia-Sastre lab; 1:10,000) with a DAPI counterstain. Infected cells (488 nM) and total cells (DAPI) were quantified using the Celigo (Nexcelcom) imaging cytometer. Infectivity was measured by the accumulation of viral NP protein in the nucleus of the Vero E6 cells (fluorescence accumulation). Percent infection was quantified as ((Infected cells/Total cells) - Background) ∗100 and the DMSO control was then set to 100% infection for analysis. The IC50 for each experiment was determined using the Prism software (GraphPad). For select inhibitors, infected supernatants were assayed for infectious viral titer using the Median Tissue Culture Infectious Dose TCID50 method. For this, infectious supernatants were collected at 48 hours post infection and frozen at −80°C until later use. Infectious titers were quantified by limiting dilution titration on Vero E6 cells. Briefly, Vero E6 cells were seeded in 96-well plates at 20,000 cells/well. The next day, SARS-CoV-2-containing supernatant was applied at serial 10-fold dilutions ranging from 10−1 to 10−6 and, after 5 days, viral CPE was detected by staining cell monolayers with crystal violet. TCID50/mL was calculated using the method of Reed and Muench.

Cytotoxicity cell viability assays
(Mount Sinai, New York). Cytotoxicity was also performed using the MTT assay (Roche), according to the manufacturer’s instructions. Cytotoxicity was performed in uninfected Vero E6 cells with same compound dilutions and concurrent with viral replication assay. All assays were performed in biologically independent triplicates.

siRNA-based knockdown of host kinases
(Institut Pasteur, Paris). Host kinases were knocked-down in A549 cells stably expressing ACE2 cells using OnTargetPlus siRNA SMARTpools (Horizon Discovery). Briefly, A549-ACE2 cells seeded at 2x104 cells/well in 96-well plates 18 hours prior to the experiment. The next day, each well was transfected with 6 pmoles of siRNA SMARTpool, using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions. Twenty-four (24) hours post transfection, the cell culture supernatant was removed and replaced with virus inoculum (MOI of 0.1 PFU/cell). Following a 1 hour adsorption at 37°C, the virus inoculum was removed and replaced with fresh Opti-MEM media (Thermo Fisher Scientific) containing 2% FBS (v/v). Seventy-two (72) hours post-infection (p.i.), the cell culture supernatant was heat-inactivated at 95°C for 5 minutes, and detection of viral genomes performed by RT-qPCR.

SARS-CoV-2 viral quantification via RT-qPCR in presence of inhibitors
(Institut Pasteur, Paris). Detection of viral genomes was performed by RT-qPCR, directly from the inactivated supernatant (Lista et al., 2020). SARS-CoV-2 specific primers targeting the N gene region: 5′-TAATCAGACAAGGAACTGATTA-3′ (Forward) and 5′-CGAAGGTGTGACTTCCATG-3′ (Reverse) (Chu et al., 2020) were used with the Luna Universal One-Step RT-qPCR Kit (NEB) in an Applied Biosystems QuantStudio 7 thermocycler, with the following cycling conditions: 55°C for 10 minutes, 95°C for 1 minute, and 40 cycles of 95°C for 10 s, followed by 60°C for 1 minute. The number of viral genomes is expressed as PFU equivalents/mL, and was calculated by performing a standard curve with RNA derived from a viral stock with a known viral titer.

SARS-CoV-2 titration by plaque assay
(Institut Pasteur, Paris). Vero E6 cells were seeded in 24-well plates at a concentration of 7.5x104 cells/well. The following day, serial dilutions were performed in serum-free MEM media. After 1 hour absorption at 37°C, 2x overlay media was added to the inoculum to give a final concentration of 2% (v/v) FBS / MEM media and 0.05% (w/v) Agarose (all Thermo Fisher Scientific) to achieve a semi-solid overlay. Plaque assays were incubated at 37°C for 3 days. Samples were fixed using 4% Formalin (Sigma Aldrich) and plaques were visualized using crystal Violet solution (Sigma Aldrich).

siRNA Cell viability assays
(Institut Pasteur, Paris). Cell viability in siRNA knocked-down cells was measured using the CellTiter Glo luminescent cell viability assay (Promega) following the manufacturer’s instructions, and luminescence measured in a Tecan Infinity 2000 plate reader. Percent viability was calculated relative to untreated cells (100% viability) and cells lysed with 20% ethanol (0% viability), included in each experiment.