A SARS-CoV-2 Protein Interaction Map Reveals Targets for Drug-Repurposing methods
Aim. Evidence-backed execution summary for A SARS-CoV-2 Protein Interaction Map Reveals Targets for Drug-Repurposing methods from A SARS-CoV-2 Protein Interaction Map Reveals Targets for Drug-Repurposing.
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human
Subject model for the experiment.
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- confirm full cohort details in the source paper
Mass spectrometry data acquisition and analysis
reagent used in the protocol.
- Use
- Samples were re-suspended 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 a Easy-nLC 1200 (Thermo Fisher Scientific) into a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) and analyzed...
Viral growth and cytotoxicity assays in the presence of inhibitors (Mt. Sinai)
reagent used in the protocol.
- Use
- 2,000 Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37C, 5% CO2. 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 prior to commencement). T...
Cells and viruses (Institut Pasteur)
reagent used in the protocol.
- Use
- African green monkey kidney epithelial Vero E6 (ATCC, CRL-1586, authenticated by ATCC and tested negative for mycoplasma contamination prior to commencement [Vero 76, clone E6, Vero E6] (ATCC® CRL-1586™)) were maintained in a humidified atmosphere at 37°C with 5% CO 2, in Dulbecco's modified E...
MAIN
reagent used in the protocol.
- Use
- So far, no clinically available antiviral drugs have been developed for SARS-CoV, SARS-CoV-2 or MERS-CoV. Clinical trials are ongoing for treatment of COVID-19 with the nucleoside analog RNA-dependent RNA Polymerase (RdRP) inhibitor Remdesivir, and recent data suggests a new nucleoside analog may be effective again...
The novel Orf10 of SARS-CoV-2 interacts with a Cullin ubiquitin ligase complex
reagent used in the protocol.
- Use
- Viruses commonly hijack ubiquitination pathways for replication and pathogenesis. The novel Orf10 of SARS-CoV-2 interacts with members of a Cullin 2 (CUL2) RING E3 ligase complex ( ), specifically the CUL2 ZYG11B complex. ZYG11B is the highest scoring protein in the Orf10 interactome, suggesting a direct interactio...
Antiviral activity of host-directed drugs and compounds
reagent used in the protocol.
- Use
- We next investigated the antiviral activity of these drugs and compounds, employing two viral assays ( ). First, at Mt Sinai Hospital in New York, we developed a medium-throughput immunofluorescence-based assay (detecting the viral NP protein) to screen 37 compounds for inhibition of SARS-CoV-2 infection in the Vero...
Antiviral activity of host-directed drugs and compounds
reagent used in the protocol.
- Use
- To better understand the mechanism by which these inhibitors exert their antiviral effects, we performed a time course assay where the drugs were added at different times relative to infection ( ). This was a single cycle infection at high MOI (2) over the course of 8 hours, where the drugs were either added 2 hours...
Antiviral activity of host-directed drugs and compounds
reagent used in the protocol.
- Use
- Coronaviruses rely on cap-dependent mRNA translation through the host translation machinery. eIF4H, an interactor of Nsp9, is a partner of eIF4A, and we observed a strong antiviral effect by the eIF4A inhibitor zotatifin ( ), which is currently in a phase I clinical trial for cancer therapy. We also observed potent...
Mass spectrometry data acquisition and analysis
Samples were re-suspended 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 a Easy-nLC 1200 (Thermo Fisher Scientific) into a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) and analyzed...
- Use
- Samples were re-suspended 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 a Easy-nLC 1200 (Thermo Fisher Scientific) into a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) and analyzed...
Viral growth and cytotoxicity assays in the presence of inhibitors (Mt. Sinai)
2,000 Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37C, 5% CO2. 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 prior to commencement). T...
- Use
- 2,000 Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37C, 5% CO2. 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 prior to commencement). T...
Affinity purification
Frozen cell pellets were thawed on ice for 15-20 minutes and suspended 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% Nonidet P 40 Substitute (NP40; Fluka Analytical) and cOmplete mini EDTA-free protease and PhosSTOP phosphatase inhibitor coc...
- Use
- Frozen cell pellets were thawed on ice for 15-20 minutes and suspended 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% Nonidet P 40 Substitute (NP40; Fluka Analytical) and cOmplete mini EDTA-free protease and PhosSTOP phosphatase inhibitor coc...
Orf6 peptide modeling
The proposed interaction between Orf6 and the NUP98-RAE1 complex was modeled in PyRosetta 4 (release v2020.02-dev61090) using the crystal structure of Vesicular stomatitis virus matrix (M) protein bound to NUP98-RAE1 as a template (PDB 4OWR downloaded from the PDB-REDO server ). The M protein chain (C) was truncated...
- Use
- The proposed interaction between Orf6 and the NUP98-RAE1 complex was modeled in PyRosetta 4 (release v2020.02-dev61090) using the crystal structure of Vesicular stomatitis virus matrix (M) protein bound to NUP98-RAE1 as a template (PDB 4OWR downloaded from the PDB-REDO server ). The M protein chain (C) was truncated...
Cell viability assays (Institut Pasteur)
Cell viability in drug-treated cells was measured using AlamarBlue reagent (ThermoFisher). Briefly, 48 h post treatment, the drug-containing media was removed and replaced with AlamarBlue and incubated for 1h at 37°C and fluorescence measured in a Tecan Infinity 2000 plate reader. Percentage viability was calcu...
- Use
- Cell viability in drug-treated cells was measured using AlamarBlue reagent (ThermoFisher). Briefly, 48 h post treatment, the drug-containing media was removed and replaced with AlamarBlue and incubated for 1h at 37°C and fluorescence measured in a Tecan Infinity 2000 plate reader. Percentage viability was calcu...
Viral growth and cytotoxicity assays in the presence of inhibitors (Mt. Sinai)
Software used for acquisition, scoring, statistics, or reporting.
- Use
- 2,000 Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37C, 5% CO2. 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 prior to commencement). T...
Gene ontology over-representation analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- The targets of each bait were tested for enrichment of Gene Ontology (GO Biological Process) terms. The over-representation analysis (ORA) was based on the hypergeometric distribution and performed using the enricher function of clusterProfiler package in R with default parameters. The gene ontology terms were obtai...
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Mass spectrometry data acquisition and analysis
Samples were re-suspended 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 a Easy-nLC 1200 (Thermo Fisher Scientific) into a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) and analyzed 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 project. All proteomic data was searched against the human proteome (uniprot reviewed sequences downloaded February 28th, 2020), EGFP sequence, and the SARS-CoV-2 protein sequences using the default settings for MaxQuant (version 1.6.11.0),. Detected peptides and proteins were filtered to 1% false disc...
Viral growth and cytotoxicity assays in the presence of inhibitors (Mt. Sinai)
2,000 Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37C, 5% CO2. 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 prior to commencement). Two hours before infection, the medium was replaced with 100ul 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 (also see Methods Institut Pasteur). Plates were then transferred into the BSL3 facility and 100 PFU (MOI 0.025) was added in 50ul of DMEM (2% FBS), bringing the final compound concentration to those indicated. Plates were then incuba...
The SARS-CoV-2 interactome reveals novel aspects of SARS-CoV-2 biology
Approximately 40% of SARS-CoV-2 interacting proteins were associated with endomembrane compartments or vesicle trafficking pathways. Host interactions of Nsp8 (signal recognition particle; SRP), Orf8 (endoplasmic reticulum protein quality control), M (ER morphology), and Nsp13 (centrosome and golgi organization) may facilitate the dramatic reconfiguration of ER/Golgi trafficking during coronavirus infection, and interactions in peripheral compartments by Nsp2 (WASH), Nsp6 and M (vacuolar ATPase), Nsp7 (Rabs), Nsp10 (AP2), E (AP3), and Orf3a (HOPS) may also modify endomembrane compartments to favor coronavirus replication. Nsp6 and Orf9c interact with Sigma receptors that are implicated in lipid remodeling and ER stress response; these proteins interact with many human drugs (see below).
Antiviral activity of host-directed drugs and compounds
To better understand the mechanism by which these inhibitors exert their antiviral effects, we performed a time course assay where the drugs were added at different times relative to infection ( ). This was a single cycle infection at high MOI (2) over the course of 8 hours, where the drugs were either added 2 hours prior to infection or at 0, 2 or 4 hours post infection. PB28, zotatifin, and hydroxychloroquine all decreased the detection of the viral NP protein even in this single cycle assay, indicating the antiviral effect occurs before viral egress from the cell ( ). Furthermore, all three molecules inhibited NP expression when added up to 4 hours post-infection, after viral entry has occurred. Thus, these molecules seem to exert their antiviral effect during viral replication.
Affinity purification
Frozen cell pellets were thawed on ice for 15-20 minutes and suspended 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% Nonidet P 40 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 minutes and partially thawed at 37°C before incubation on a tube rotator for 30 minutes at 4°C and centrifugation at 13,000 × g, 4°C for 15 minutes 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 supple...
Transfection
For each affinity purification (26 wild-type and one catalytically dead SARS-CoV-2 baits, one GFP control, one empty vector control), ten million HEK293T/17 cells were plated per 15-cm dish and transfected with up to 15 µg of individual Strep-tagged expression constructs after 20-24 hours. 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 to transfection reagent based on manufacturer's recommendations. After more than 38 hours, cells were dissociated at room temperature using 10 ml Dulbecco's Phosphate Buffered Saline without calcium and magnesium (D-PBS) supplemented with 10 mM EDTA for at least 5 minutes and subsequently washed with 10 ml D-PBS. Each step was followed by centrifugation at 200 × g, 4°C for 5 minutes. Cell pellet...
On-bead digestion
Bead-bound proteins were denatured and reduced at 37°C for 30 minutes and after bringing to room temperature, alkylated in the dark with 3 mM iodoacetamide for 45 minutes and quenched with 3 mM DTT for 10 minutes. Proteins were then incubated at 37°C, initially for 4 hours with 1.5 µl trypsin (0.5 µg/µl; Promega) and then another 1-2 hours 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 (20mg PROTO 300 C18; The Nest Group, Inc.) according to stan...
Antiviral activity assays (Institut Pasteur)
Vero E6 cells were seeded at 1.5 × 10 4 cells per well in 96-well plates 18h prior to the experiment. Two hours prior to infection, the cell culture supernatant of triplicate wells was replaced with media 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 media was removed, and replaced with virus inoculum (MOI of 0.1 PFU/cell) containing TPCK-trypsin (Sigma-Aldrich). Following a one-hour adsorption at 37 °C, the virus inoculum was removed and 200µL of drug- (or vehicle-) containing media added. 48h post infection (p.i.), 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 perfor...
Measurement outputs
What raw and processed outputs should exist?
Samples were re-suspended in 4% formic acid, 2% acetonitrile solution, and separated by a reversed-phase gradient over a nanoflow C18 column (Dr. Maisch). Each sample was direct...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
2,000 Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37C, 5% CO2. Vero E6 cells used were purchased from ATCC and thus authenticated (...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Sequence analysis of SARS-CoV-2 isolates suggests that the 30kb genome encodes as many as 14 open reading frames (Orfs). The Orf1a / Orf1ab encodes a polyprotein, which is auto-...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Mature Nsps, with the exception of Nsp3 and Nsp16, and all predicted proteins expressed from other SARS-CoV-2 Orfs (27 proteins plus one mutant) were codon optimized and cloned...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
2,000 Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37C, 5% CO2.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Samples were re-suspended in 4% formic acid, 2% acetonitrile solution, and separated by a reversed-phase gradient over a nanoflow C18 column (Dr. Maisch). Each sample was direct...; 2,000 Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37C, 5% CO2. Vero E6 cells used were purchased from ATCC and thus authenticated (...; Sequence analysis of SARS-CoV-2 isolates suggests that the 30kb genome encodes as many as 14 open reading frames (Orfs). The Orf1a / Orf1ab encodes a polyprotein, which is auto-...; Mature Nsps, with the exception of Nsp3 and Nsp16, and all predicted proteins expressed from other SARS-CoV-2 Orfs (27 proteins plus one mutant) were codon optimized and cloned....
from paperStatistical comparison
2,000 Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37C, 5% CO2. Vero E6 cells used were purchased from ATCC and thus authenticated (...; While the cell line used for these AP-MS experiments, HEK-293T/17, is permissive to SARS-CoV-2 infection, it does not represent the primary physiological site of infection, lun...; To disrupt the SARS-CoV-2 interactome, we sought ligands of the human interacting proteins (Methods). Molecules were prioritized by the statistical significance of the interacti...; The targets of each bait were tested for enrichment of Gene Ontology (GO Biological Process) terms. The over-representation analysis (ORA) was based on the hypergeometric distri...
from paperReporting output
Report representative outputs alongside summary comparisons for Samples were re-suspended in 4% formic acid, 2% acetonitrile solution, and separated by a reversed-phase gradient over a nanoflow C18 column (Dr. Maisch). Each sample was direct..., 2,000 Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37C, 5% CO2. Vero E6 cells used were purchased from ATCC and thus authenticated (..., Sequence analysis of SARS-CoV-2 isolates suggests that the 30kb genome encodes as many as 14 open reading frames (Orfs). The Orf1a / Orf1ab encodes a polyprotein, which is auto-..., Mature Nsps, with the exception of Nsp3 and Nsp16, and all predicted proteins expressed from other SARS-CoV-2 Orfs (27 proteins plus one mutant) were codon optimized and cloned....
inferred from protocolStructured statistical methods
2,000 Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37C, 5% CO2. Vero E6 cells used were purchased from ATCC and thus authenticated (...; While the cell line used for these AP-MS experiments, HEK-293T/17, is permissive to SARS-CoV-2 infection, it does not represent the primary physiological site of infection, lun...; To disrupt the SARS-CoV-2 interactome, we sought ligands of the human interacting proteins (Methods). Molecules were prioritized by the statistical significance of the interacti...; The targets of each bait were tested for enrichment of Gene Ontology (GO Biological Process) terms. The over-representation analysis (ORA) was based on the hypergeometric distri...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Samples were re-suspended 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 a Easy-nLC 1200 (Thermo Fisher Scientific) into a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) and analyzed 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 project. All proteomic data was searched against the human proteome (uniprot reviewed sequences downloaded February 28th, 2020), EGFP sequence, and the SARS-CoV-2 protein sequences using the default settings for MaxQuant (version 1.6.11.0),. 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 (version 3.6.3) and MiST ( https://github.com/kroganlab/mist ),. We applied a two-step filtering strategy to determine the final list of reported interactors which reli...
2,000 Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37C, 5% CO2. 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 prior to commencement). Two hours before infection, the medium was replaced with 100ul 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 (also see Methods Institut Pasteur). Plates were then transferred into the BSL3 facility and 100 PFU (MOI 0.025) was added in 50ul of DMEM (2% FBS), bringing the final compound concentration to those indicated. Plates were then incubated for 48 h at 37C. After infection, supernatants were removed and cells were fixed with 4% formaldehyde for 24 hours prior to being removed from the BSL3 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 counter...
Approximately 40% of SARS-CoV-2 interacting proteins were associated with endomembrane compartments or vesicle trafficking pathways. Host interactions of Nsp8 (signal recognition particle; SRP), Orf8 (endoplasmic reticulum protein quality control), M (ER morphology), and Nsp13 (centrosome and golgi organization) may facilitate the dramatic reconfiguration of ER/Golgi trafficking during coronavirus infection, and interactions in peripheral compartments by Nsp2 (WASH), Nsp6 and M (vacuolar ATPase), Nsp7 (Rabs), Nsp10 (AP2), E (AP3), and Orf3a (HOPS) may also modify endomembrane compartments to favor coronavirus replication. Nsp6 and Orf9c interact with Sigma receptors that are implicated in lipid remodeling and ER stress response; these proteins interact with many human drugs (see below).
To better understand the mechanism by which these inhibitors exert their antiviral effects, we performed a time course assay where the drugs were added at different times relative to infection ( ). This was a single cycle infection at high MOI (2) over the course of 8 hours, where the drugs were either added 2 hours prior to infection or at 0, 2 or 4 hours post infection. PB28, zotatifin, and hydroxychloroquine all decreased the detection of the viral NP protein even in this single cycle assay, indicating the antiviral effect occurs before viral egress from the cell ( ). Furthermore, all three molecules inhibited NP expression when added up to 4 hours post-infection, after viral entry has occurred. Thus, these molecules seem to exert their antiviral effect during viral replication.
Frozen cell pellets were thawed on ice for 15-20 minutes and suspended 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% Nonidet P 40 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 minutes and partially thawed at 37°C before incubation on a tube rotator for 30 minutes at 4°C and centrifugation at 13,000 × g, 4°C for 15 minutes 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 hours. 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 b...
For each affinity purification (26 wild-type and one catalytically dead SARS-CoV-2 baits, one GFP control, one empty vector control), ten million HEK293T/17 cells were plated per 15-cm dish and transfected with up to 15 µg of individual Strep-tagged expression constructs after 20-24 hours. 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 to transfection reagent based on manufacturer's recommendations. After more than 38 hours, cells were dissociated at room temperature using 10 ml Dulbecco's Phosphate Buffered Saline without calcium and magnesium (D-PBS) supplemented with 10 mM EDTA for at least 5 minutes and subsequently washed with 10 ml D-PBS. Each step was followed by centrifugation at 200 × g, 4°C for 5 minutes. 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.
Bead-bound proteins were denatured and reduced at 37°C for 30 minutes and after bringing to room temperature, alkylated in the dark with 3 mM iodoacetamide for 45 minutes and quenched with 3 mM DTT for 10 minutes. Proteins were then incubated at 37°C, initially for 4 hours with 1.5 µl trypsin (0.5 µg/µl; Promega) and then another 1-2 hours 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 (20mg PROTO 300 C18; The Nest Group, Inc.) according to standard protocols.
Vero E6 cells were seeded at 1.5 × 10 4 cells per well in 96-well plates 18h prior to the experiment. Two hours prior to infection, the cell culture supernatant of triplicate wells was replaced with media 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 media was removed, and replaced with virus inoculum (MOI of 0.1 PFU/cell) containing TPCK-trypsin (Sigma-Aldrich). Following a one-hour adsorption at 37 °C, the virus inoculum was removed and 200µL of drug- (or vehicle-) containing media added. 48h post infection (p.i.), 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 primers. 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 (NE...
Machine-readable layer
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"name": "A SARS-CoV-2 Protein Interaction Map Reveals Targets for Drug-Repurposing methods",
"description": "Evidence-backed execution summary for A SARS-CoV-2 Protein Interaction Map Reveals Targets for Drug-Repurposing methods from A SARS-CoV-2 Protein Interaction Map Reveals Targets for Drug-Repurposing.",
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{
"@type": "HowToStep",
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"name": "Mass spectrometry data acquisition and analysis",
"text": "Samples were re-suspended 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 a Easy-nLC 1200 (Thermo Fisher Scientific) into a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) and analyzed 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 project. All proteomic data was searched against the human proteome (uniprot reviewed sequences downloaded February 28th, 2020), EGFP sequence, and the SARS-CoV-2 protein sequences using the default settings for MaxQuant (version 1.6.11.0),. Detected peptides and proteins were filtered to 1% false disc..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Viral growth and cytotoxicity assays in the presence of inhibitors (Mt. Sinai)",
"text": "2,000 Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37C, 5% CO2. 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 prior to commencement). Two hours before infection, the medium was replaced with 100ul 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 (also see Methods Institut Pasteur). Plates were then transferred into the BSL3 facility and 100 PFU (MOI 0.025) was added in 50ul of DMEM (2% FBS), bringing the final compound concentration to those indicated. Plates were then incuba..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "The SARS-CoV-2 interactome reveals novel aspects of SARS-CoV-2 biology",
"text": "Approximately 40% of SARS-CoV-2 interacting proteins were associated with endomembrane compartments or vesicle trafficking pathways. Host interactions of Nsp8 (signal recognition particle; SRP), Orf8 (endoplasmic reticulum protein quality control), M (ER morphology), and Nsp13 (centrosome and golgi organization) may facilitate the dramatic reconfiguration of ER/Golgi trafficking during coronavirus infection, and interactions in peripheral compartments by Nsp2 (WASH), Nsp6 and M (vacuolar ATPase), Nsp7 (Rabs), Nsp10 (AP2), E (AP3), and Orf3a (HOPS) may also modify endomembrane compartments to favor coronavirus replication. Nsp6 and Orf9c interact with Sigma receptors that are implicated in lipid remodeling and ER stress response; these proteins interact with many human drugs (see below)."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Antiviral activity of host-directed drugs and compounds",
"text": "To better understand the mechanism by which these inhibitors exert their antiviral effects, we performed a time course assay where the drugs were added at different times relative to infection ( ). This was a single cycle infection at high MOI (2) over the course of 8 hours, where the drugs were either added 2 hours prior to infection or at 0, 2 or 4 hours post infection. PB28, zotatifin, and hydroxychloroquine all decreased the detection of the viral NP protein even in this single cycle assay, indicating the antiviral effect occurs before viral egress from the cell ( ). Furthermore, all three molecules inhibited NP expression when added up to 4 hours post-infection, after viral entry has occurred. Thus, these molecules seem to exert their antiviral effect during viral replication."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Affinity purification",
"text": "Frozen cell pellets were thawed on ice for 15-20 minutes and suspended 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% Nonidet P 40 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 minutes and partially thawed at 37°C before incubation on a tube rotator for 30 minutes at 4°C and centrifugation at 13,000 × g, 4°C for 15 minutes 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 supple..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Transfection",
"text": "For each affinity purification (26 wild-type and one catalytically dead SARS-CoV-2 baits, one GFP control, one empty vector control), ten million HEK293T/17 cells were plated per 15-cm dish and transfected with up to 15 µg of individual Strep-tagged expression constructs after 20-24 hours. 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 to transfection reagent based on manufacturer's recommendations. After more than 38 hours, cells were dissociated at room temperature using 10 ml Dulbecco's Phosphate Buffered Saline without calcium and magnesium (D-PBS) supplemented with 10 mM EDTA for at least 5 minutes and subsequently washed with 10 ml D-PBS. Each step was followed by centrifugation at 200 × g, 4°C for 5 minutes. Cell pellet..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "On-bead digestion",
"text": "Bead-bound proteins were denatured and reduced at 37°C for 30 minutes and after bringing to room temperature, alkylated in the dark with 3 mM iodoacetamide for 45 minutes and quenched with 3 mM DTT for 10 minutes. Proteins were then incubated at 37°C, initially for 4 hours with 1.5 µl trypsin (0.5 µg/µl; Promega) and then another 1-2 hours 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 (20mg PROTO 300 C18; The Nest Group, Inc.) according to stan..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Antiviral activity assays (Institut Pasteur)",
"text": "Vero E6 cells were seeded at 1.5 × 10 4 cells per well in 96-well plates 18h prior to the experiment. Two hours prior to infection, the cell culture supernatant of triplicate wells was replaced with media 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 media was removed, and replaced with virus inoculum (MOI of 0.1 PFU/cell) containing TPCK-trypsin (Sigma-Aldrich). Following a one-hour adsorption at 37 °C, the virus inoculum was removed and 200µL of drug- (or vehicle-) containing media added. 48h post infection (p.i.), 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 perfor..."
}
],
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{
"@type": "HowToTool",
"name": "Mass spectrometry data acquisition and analysis"
},
{
"@type": "HowToTool",
"name": "Viral growth and cytotoxicity assays in the presence of inhibitors (Mt. Sinai)"
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{
"@type": "HowToTool",
"name": "Affinity purification"
},
{
"@type": "HowToTool",
"name": "Orf6 peptide modeling"
},
{
"@type": "HowToTool",
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{
"@type": "HowToSupply",
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{
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{
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{
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"name": "The novel Orf10 of SARS-CoV-2 interacts with a Cullin ubiquitin ligase complex"
},
{
"@type": "HowToSupply",
"name": "Antiviral activity of host-directed drugs and compounds"
},
{
"@type": "HowToSupply",
"name": "Antiviral activity of host-directed drugs and compounds"
},
{
"@type": "HowToSupply",
"name": "Antiviral activity of host-directed drugs and compounds"
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