Genome annotation

The GenBank sequence for SARS-CoV-2 isolate 2019-nCoV/USA-WA1/2020, accession MN985325, was downloaded on 24 January 2020. In total, we annotated 29 possible ORFs and proteolytically mature proteins encoded by SARS-CoV-21,12. Proteolytic products that result from NSP3- and NSP5-mediated cleavage of the ORF1a/ORF1ab polyprotein were predicted on the basis of the protease specificity of SARS-CoV proteases56, and 16 predicted nonstructural proteins (NSPs) were subsequently cloned (NSP1–NSP16). For the NSP5 protease (3Clike/3CLpro), we also designed the catalytically dead mutant NSP5(C145A)57,58. ORFs at the 3′ end of the viral genome annotated in the original GenBank file included 4 structural proteins: S, E, M, N and the additional ORFs ORF3a, ORF6, ORF7a, ORF8 and ORF10. On the basis of the analysis of ORFs in the genome and comparisons with other annotated SARS-CoV ORFs, we annotated a further four ORFs: ORF3b, ORF7b, ORF9b and ORF9c.

Cloning

ORFs and proteolytically mature NSPs annotated in the SARS-CoV-2 genome were human codon-optimized using the IDT codon-optimization tool (https://www.idtdna.com/codonopt) and internal EcoRI and BamHI sites were eliminated. Start and stop codons were added as necessary to NSPs 1–16, a Kozak sequence was added before each start codon, and a 2×Strep tag with linker was added to either the N or C terminus. To guide our tagging strategy, we used GPS-Lipid to predict protein lipid modification of the termini (http://lipid.biocuckoo.org/webserver.php)59,60, TMHMM Server v.2.0 to predict transmembrane/hydrophobic regions (http://www.cbs.dtu.dk/services/TMHMM/)61 and SignalP v.5.0 to predict signal peptides (http://www.cbs.dtu.dk/services/SignalP/)62. IDT gBlocks were ordered for all reading frames with 15-bp overlaps that corresponded to flanking sequences of the EcoRI and BamHI restriction sites in the lentiviral constitutive expression vector pLVX-EF1alpha-IRES-Puro (Takara). Vectors were digested and gel-purified, and gene fragments were cloned using InFusion (Takara). The S protein was synthesized and cloned into pTwist-EF1alpha-IRES-Puro (Twist Biosciences). NSP16 showed multiple mutations that could not be repaired before the time-sensitive preparation of this manuscript, and NSP3 was too large to be synthesized in time to be included in this study. Strep-tagged constructs encoding NSP3, NSP3(C857A) (catalytically dead mutant) and NSP16 will be used in future AP-MS experiments.

Cell culture

HEK-293T/17 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Corning) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies) and 1% penicillin–streptomycin (Corning) and maintained at 37 °C in a humidified atmosphere of 5% CO 2 . HEK-293T/17 cells were procured from the UCSF Cell Culture Facility, now available through UCSF’s Cell and Genome Engineering Core ((https://cgec.ucsf.edu/cell-culture-and-banking-services); cell line collection listed here: https://ucsf.app.box.com/s/6xkydeqhr8a2xes0mbo2333i3k1lndqv (CCLZR076)). STR analysis by the Berkeley Cell Culture Facility on 8 August 2017 authenticated HEK-293T/17 cells with 94% probability. Cells were tested on 3 July 2019 using the MycoAlert Mycoplasma Detection Kit (Lonza LT07-318) and were negative: B/A ratio < 1 (no detected mycoplasma).

Transfection

For each affinity purification (26 wild-type baits and one catalytically dead SARS-CoV-2 bait, one GFP control and one empty vector control), ten million HEK-293T/17 cells were plated per 15-cm dish and transfected with up to 15 μg of individual Strep-tagged expression constructs after 20–24 h. Total plasmid was normalized to 15 μg with empty vector and complexed with PolyJet Transfection Reagent (SignaGen Laboratories) at a 1:3 μg:μl ratio of plasmid:transfection reagent based on the manufacturer’s recommendations. After more than 38 h, cells were dissociated at room temperature using 10 ml Dulbecco’s phosphate-buffered saline without calcium and magnesium (DPBS) supplemented with 10 mM EDTA for at least 5 min and subsequently washed with 10 ml DPBS. Each step was followed by centrifugation at 200g, 4 °C for 5 min. Cell pellets were frozen on dry ice and stored at −80 °C. For each bait, n = 3 independent biological replicates were prepared for affinity purification.

Affinity purification

Frozen cell pellets were thawed on ice for 15–20 min and resuspended in 1 ml lysis buffer (IP buffer (50 mM Tris-HCl, pH 7.4 at 4 °C, 150 mM NaCl, 1 mM EDTA) supplemented with 0.5% Non-idet P40 substitute (NP40; Fluka Analytical) and cOmplete mini EDTA-free protease and PhosSTOP phosphatase inhibitor cocktails (Roche)). Samples were then frozen on dry ice for 10–20 min and partially thawed at 37 °C before incubation on a tube rotator for 30 min at 4 °C and centrifugation at 13,000g, 4 °C for 15 min to pellet debris. After reserving 50 μl lysate, up to 48 samples were arrayed into a 96-well Deepwell plate for affinity purification on the KingFisher Flex Purification System (Thermo Scientific) as follows: MagStrep ‘type3’ beads (30 μl; IBA Lifesciences) were equilibrated twice with 1 ml wash buffer (IP buffer supplemented with 0.05% NP40) and incubated with 0.95 ml lysate for 2 h. Beads were washed three times with 1 ml wash buffer and then once with 1 ml IP buffer. To directly digest bead-bound proteins as well as elute proteins with biotin, beads were manually suspended in IP buffer and divided in half before transferring to 50 μl denaturation–reduction buffer (2 M urea, 50 mM Tris-HCl pH 8.0, 1 mM DTT) and 50 μl 1× buffer BXT (IBA Lifesciences) dispensed into a single 96-well KF microtitre plate. Purified proteins were first eluted at room temperature for 30 min with constant shaking at 1,100 rpm on a ThermoMixer C incubator. After removing eluates, on-bead digestion proceeded (see ‘On-bead digestion’). Strep-tagged protein expression in lysates and enrichment in eluates were assessed by western blot and silver stain, respectively. The KingFisher Flex Purification System was placed in the cold room and allowed to equilibrate to 4 °C overnight before use. All automated protocol steps were performed using the slow mix speed and the following mix times: 30 s for equilibration and wash steps, 2 h for binding and 1 min for final bead release. Three 10-s bead collection times were used between all steps.

On-bead digestion

Bead-bound proteins were denatured and reduced at 37 °C for 30 min and after being brought to room temperature, alkylated in the dark with 3 mM iodoacetamide for 45 min and quenched with 3 mM DTT for 10 min. Proteins were then incubated at 37 °C, initially for 4 h with 1.5 μl trypsin (0.5 μg/μl; Promega) and then another 1–2 h with 0.5 μl additional trypsin. To offset evaporation, 15 μl 50 mM Tris-HCl, pH 8.0 were added before trypsin digestion. All steps were performed with constant shaking at 1,100 rpm on a ThermoMixer C incubator. Resulting peptides were combined with 50 μl 50 mM Tris-HCl, pH 8.0 used to rinse beads and acidified with trifluoroacetic acid (0.5% final, pH < 2.0). Acidified peptides were desalted for MS analysis using a BioPureSPE Mini 96-Well Plate (20 mg PROTO 300 C18; The Nest Group) according to standard protocols.

MS data acquisition and analysis

Samples were resuspended in 4% formic acid, 2% acetonitrile solution, and separated by a reversed-phase gradient over a Nanoflow C18 column (Dr Maisch). Each sample was directly injected via an Easy-nLC 1200 (Thermo Fisher Scientific) into a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) and analysed with a 75 min acquisition, with all MS1 and MS2 spectra collected in the orbitrap; data were acquired using the Thermo software Xcalibur (4.2.47) and Tune (2.11 QF1 Build 3006). For all acquisitions, QCloud was used to control instrument longitudinal performance during the project63. All proteomic data were searched against the human proteome (Uniprot-reviewed sequences downloaded 28 February 2020), the eGFP sequence and the SARS-CoV-2 protein sequences using the default settings for MaxQuant (v.1.6.11.0)64,65. Detected peptides and proteins were filtered to 1% false-discovery rate in MaxQuant, and identified proteins were then subjected to protein–protein interaction scoring with both SAINTexpress (v.3.6.3)14 and MiST (https://github.com/kroganlab/mist)15,66. We applied a two-step filtering strategy to determine the final list of reported interactors, which relied on two different scoring stringency cut-offs. In the first step, we chose all protein interactions that had a MiST score ≥ 0.7, a SAINTexpress Bayesian false-discovery rate (BFDR) ≤ 0.05 and an average spectral count ≥ 2. For all proteins that fulfilled these criteria, we extracted information about the stable protein complexes that they participated in from the CORUM67 database of known protein complexes. In the second step, we then relaxed the stringency and recovered additional interactors that (1) formed complexes with interactors determined in filtering step 1 and (2) fulfilled the following criteria: MiST score ≥ 0.6, SAINTexpress BFDR ≤ 0.05 and average spectral counts ≥ 2. Proteins that fulfilled filtering criteria in either step 1 or step 2 were considered to be high-confidence protein–protein interactions (HC-PPIs) and visualized with Cytoscape (v.3.7.1)68. Using this filtering criteria, nearly all of our baits recovered a number of HC-PPIs in close alignment with previous datasets reporting an average of around 6 PPIs per bait69. However, for a subset of baits (ORF8, NSP8, NSP13 and ORF9c), we observed a much higher number of PPIs that passed these filtering criteria. For these four baits, the MiST scoring was instead performed using a larger in-house database of 87 baits that were prepared and processed in an analogous manner to this SARS-CoV-2 dataset. This was done to provide a more comprehensive collection of baits for comparison, to minimize the classification of non-specifically binding background proteins as HC-PPIs. All MS raw data and search results files have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD01811770,71. PPI networks have also been uploaded to NDEx.

GO overrepresentation analysis

The targets of each bait were tested for enrichment of GO biological process terms. The overrepresentation analysis was based on the hypergeometric distribution and performed using the enricher function of clusterProfiler package in R with default parameters. The GO terms were obtained from the c5 category of Molecular Signature Database (MSigDBv6.1). Significant GO terms (1% false-discovery rate) were identified and further refined to select non-redundant terms. To select non-redundant gene sets, we first constructed a GO term tree based on distances (1 − Jaccard similarity coefficients of shared genes) between the significant terms. The GO term tree was cut at a specific level (h = 0.99) to identify clusters of non-redundant gene sets. For a bait with multiple significant terms belonging to the same cluster, we selected the broadest term that is, largest gene set size.

Virus interactome similarity analysis

Interactome similarity was assessed by comparing the number of shared human-interacting proteins between pathogen pairs, using a hypergeometric test to calculate significance. The background gene set for the test consisted of all unique proteins detected by MS across all pathogens (n = 10,181 genes).

ORF6 peptide modelling

The proposed interaction between ORF6 and the NUP98–RAE1 complex was modelled in PyRosetta 4 (release v.2020.02-dev61090)72 using the crystal structure of VSV matrix (M) protein bound to NUP98–RAE1 as a template32 (PDB 4OWR; downloaded from the PDB-REDO server73). The M protein chain (C) was truncated after residue 54 to restrict the model to the putative interaction motif in ORF6 (M protein residues 49–54, sequence DEMDTH). These residues were mutated to the ORF6 sequence, QPMEID, using the mutate_residue function in the module pyrosetta.toolbox, without repacking at this initial step. After all six residues were mutated, the full model was relaxed to a low-energy conformation using the FastRelax protocol in the module pyrosetta.rosetta.protocols.relax. FastRelax was run with constraints to starting coordinates and scored with the ref2015 score function. The resulting model was inspected for any large energetic penalties associated with the modelled peptide residues or those NUP98 and RAE1 residues interacting with the peptide, and was found to have none. The model was visualized in PyMOL (The PyMOL Molecular Graphics System, v.3.4, Schrödinger).

ORF10 secondary structure prediction

The secondary structure of ORF10 was predicted using JPRED (https://www.compbio.dundee.ac.uk/jpred/index.html)74.

Protein E alignment

Protein E sequences from SARS-CoV-2 (YP_009724392.1), SARS-CoV (NP_828854.1) and bat SARS-like CoV (AGZ48809.1) were aligned using Clustal Omega75, and then manually aligned to the sequences of histone H3 (P68431) and influenza A H3N2 NS1 (YP_308845.1).

Chemoinformatic analysis of SARS-CoV-2-interacting partners

To identify drugs and reagents that modulate the 332 host factors that interact with SARS-CoV-2 and HEK-293T/17 cells (MiST ≥ 0.70), we used two approaches: (1) a chemoinformatic analysis of open-source chemical databases and (2) a target- and pathway-specific literature search, drawing on specialist knowledge within our group. Chemoinformatically, we retrieved 2,472 molecules from the IUPHAR/BPS Guide to Pharmacology (2020-3-12)55 (Supplementary Table 7) that interacted with 30 human ‘prey’ proteins (38 approved, 71 in clinical trials), and found 10,883 molecules (95 approved, 369 in clinical trials) from the ChEMBL25 database76 (Supplementary Table 8). For both approaches, molecules were prioritized on their FDA approval status, activity at the target of interest better than 1 μM and commercial availability, drawing on the ZINC database77. FDA-approved molecules were prioritized except when clinical candidates or preclinical research molecules had substantially better selectivity or potency on-target. In some cases, we considered molecules with indirect mechanisms of action on the general pathway of interest based solely on literature evidence (for example, captopril modulates ACE2 indirectly via its direct interaction with angiotensin-converting enzyme, ACE). Finally, we predicted 6 additional molecules (2 approved, 1 in clinical trials) for proteins with MIST scores between 0.7 and 0.6 to viral baits (Supplementary Tables 4, 5). Complete methods can be found at https://github.com/momeara/BioChemPantry/tree/master/vignette/COVID19.

Molecular docking

After their chemoinformatic assignment to the sigma-1 receptor, cloperastine and clemastine were docked into the agonist-bound state structure of the receptor (6DK1)78 using DOCK3.779. The best scoring configurations that ion pair with Glu172 are shown; both l-cloperastine and clemastine receive solvation-corrected docking scores between −42 and −43 kcal/mol, indicating high complementarity.

Viral growth and cytotoxicity assays in the presence of inhibitors

For studies carried out at Mount Sinai, SARS-CoV-2 (isolate USA-WA1/2020 from BEI RESOURCES NR-52281) was propagated in Vero E6 cells. Two thousand Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37 °C, 5% CO 2 . Vero E6 cells used were purchased from ATCC and thus authenticated (VERO C1008 (Vero 76, clone E6, Vero E6) (ATCC CRL-1586); tested negative for mycoplasma contamination before commencement). Then, 2 h 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. The Vero E6 cell line used in this study is a kidney cell line; therefore, we cannot exclude that lung cells yield different results for some inhibitors (see also ‘Cells and viruses’, ‘Antiviral activity assays’, ‘Cell viability assays’ and ‘Plaque-forming assays’ for studies carried out at Institut Pasteur). Plates were then transferred into the Biosafety Level 3 (BSL3) facility and 100 PFU (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 h at 37 °C. After infection, supernatants were removed and cells were fixed with 4% formaldehyde for 24 h before being removed from the BSL3 facility. The cells were then immunostained for the viral NP protein (anti-sera produced in the García -Sastre laboratory; 1:10,000) with a DAPI counterstain. Infected cells (488 nM) and total cells (DAPI) were quantified using the Celigo (Nexcelcom) imaging cytometer. Infectivity is measured by the accumulation of viral NP protein in the nucleus of the Vero E6 cells (fluorescence accumulation). Percentage infection was quantified as ((infected cells/total cells) − background) × 100 and the DMSO control was then set to 100% infection for analysis. The IC 50 and IC 90 for each experiment were determined using the Prism (GraphPad) software. For some inhibitors, infected supernatants were assayed for infectious viral titres using the TCID 50 method. For this, infectious supernatants were collected at 48 h after infection and frozen at −80 °C until later use. Infectious titres were quantified by limiting dilution titration on Vero E6 cells. In brief, Vero E6 cells were seeded in 96-well plates at 20,000 cells per well. The next day, SARS-CoV-2-containing supernatant was applied at serial tenfold dilutions ranging from 10−1 to 10−6 and, after 5 days, viral cytopathogenic effect was detected by staining cell monolayers with crystal violet. The TCID 50 /ml values were calculated using the previously described method80. Cytotoxicity was also performed using the MTT assay (Roche), according to the manufacturer’s instructions. Cytotoxicity was performed in uninfected VeroE6 cells with same compound dilutions and concurrent with viral replication assay. All assays were performed in biologically independent triplicates.

Cells and viruses

For studies at the Institut Pasteur, African green monkey kidney epithelial Vero E6 cells (ATCC, CRL-1586, authenticated by ATCC and tested negative for mycoplasma contamination before commencement (Vero 76, clone E6, Vero E6) (ATCC CRL-1586)) were maintained in a humidified atmosphere at 37 °C with 5% CO 2 , in DMEM containing 10% (v/v) FBS (Invitrogen) and 5 units/ml penicillin and 5 μg/ml streptomycin (Life Technologies). The Vero E6 cell line used in this study is a kidney cell line; therefore, we cannot exclude that lung cells yield different results for some inhibitors (see also ‘Viral growth and cytotoxicity assays in the presence of inhibitors’ for studies performed at Mount Sinai). SARS-CoV-2, isolate France/IDF0372/2020, was supplied by the National Reference Centre for Respiratory Viruses hosted by the Institut Pasteur and headed by S. van der Werf. The human sample from which strain BetaCoV/France/IDF0372/2020 was isolated has been provided by X. Lescure and Y. Yazdanpanah from the Bichat Hospital, Paris, France. The BetaCoV/France/IDF0372/2020 strain was supplied through the European Virus Archive goes Global (Evag) platform, a project that has received funding from the European Union’s Horizon 2020 research and innovation programme under the grant agreement no. 653316. Viral stocks were prepared by propagation in Vero E6 cells in DMEM supplemented with 2% FBS and 1 μg/ml TPCK-trypsin (Sigma-Aldrich). Viral titres were determined by plaque-forming assay in minimum essential medium supplemented with 2% (v/v) FBS (Invitrogen) and 0.05% agarose. All experiments involving live SARS-CoV-2 were performed at the Institut Pasteur in compliance with the guidelines of the Institut Pasteur following BSL3 containment procedures in approved laboratories. All experiments were performed in at least three biologically independent samples.

Antiviral activity assays

Vero E6 cells were seeded at 1.5 × 104 cells per well in 96-well plates 18 h before the experiment. Then, 2 h before infection, the cell-culture supernatant of triplicate wells was replaced with medium containing 10 μM, 2 μM, 500 nM, 200 nM, 100 nM or 10 nM of each compound or the equivalent volume of maximum DMSO vehicle used as a control. At the time of infection, the drug-containing medium was removed, and replaced with virus inoculum (MOI of 0.1 PFU per cell) containing TPCK-trypsin (Sigma-Aldrich). Following a 1-h adsorption incubation at 37 °C, the virus inoculum was removed and 200 μl of drug- or vehicle-containing medium was added. Then, 48 h after infection, the cell-culture supernatant was used to extract RNA using the Direct-zol-96 RNA extraction kit (Zymo) following the manufacturer’s instructions. Detection of viral genomes in the extracted RNA was performed by RT–qPCR, using previously published SARS-CoV-2-specific primers81. Specifically, the primers target the N gene region: 5′-TAATCAGACAAGGAACTGATTA-3′ (forward) and 5′-CGAAGGTGTGACTTCCATG-3′ (reverse). RT–qPCR was performed using the Luna Universal One-Step RT–qPCR Kit (NEB) in an Applied Biosystems QuantStudio 6 thermocycler, using the following cycling conditions: 55 °C for 10 min, 95 °C for 1 min, and 40 cycles of 95 °C for 10 s, followed by 60 °C for 1 min. The quantity of viral genomes is expressed as PFU equivalents, and was calculated by performing a standard curve with RNA derived from a viral stock with a known viral titre. In addition to measuring viral RNA in the supernatant derived from drug-treated cells, infectious virus was quantified by plaque-forming assay.

Cell viability assays

Cell viability in drug-treated cells was measured using Alamar blue reagent (ThermoFisher). In brief, 48 h after treatment, the drug-containing medium was removed and replaced with Alamar blue and incubated for 1 h at 37 °C and fluorescence measured in a Tecan Infinity 2000 plate reader. Percentage viability was calculated relative to untreated cells (100% viability) and cells lysed with 20% ethanol (0% viability), included in each plate.

Plaque-forming assays

Viruses were quantified by plaque-forming assays. For this, Vero E6 cells were seeded in 24-well plates at a concentration of 7.5 × 104 cells per well. The following day, tenfold serial dilutions of individual virus samples in serum-free MEM medium were added to infect the cells at 37 °C for 1 h. After the adsorption time, the overlay medium was added at final concentration of 2% FBS/MEM medium and 0.05% agarose to achieve a semi-solid overlay. Plaque-forming assays were incubated at 37 °C for 3 days before fixation with 4% formalin and visualization using crystal violet solution.

Off-target assays for Sigma receptor drugs and ligands

hERG binding assays were carried out as previously described82. In brief, compounds were incubated with hERG membranes, prepared from HEK-293 cells stably expressing hERG channels, and [3H]dofetilide (5 nM final) in a total of 150 μl for 90 min at room temperature in the dark. Reactions were stopped by filtering the mixture onto a glass fibre and were quickly washed three times to remove unbound [3H]dofetilide. The filter was dried in a microwave, melted with a scintillant cocktail and wrapped in a plastic film. Radioactivity was counted on a MicroBeta counter and results were analysed in Prism by fitting to the built-in one binding function to obtain affinity K i . Radioligand binding assays for the muscarinic and alpha-adrenergic receptors were performed as previously described83. Detailed protocols are available on the NIMH PDSP website at https://pdspdb.unc.edu/html/tutorials/UNC-CH%20Protocol%20Book.pdf.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.