Large scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities
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Abstract
Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, most current large-scale methods do not identify this important class of protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a viral peptide discovery approach covering 23 coronavirus strains that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an ΦxFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its ΦxFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction dampened SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents.
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SciScore for 10.1101/2021.04.19.440086: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization Number of cells highly infected (strong N staining) was counted in 12 random fields (magnification 20x) and % of cells expressing stress granule (G3BP1) signals in these were calculated. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Virus was detected using primary monoclonal rabbit antibodies directed against SARS-CoV-2 nucleocapsid (Sino Biological Inc., 40143-R001), and secondary anti-rabbit HRP conjugated antibodies (1:2000, Thermo Fisher Scientific). SARS-CoV-2 nucleocapsid ( Sino Biological Inc . , 40143-R001) ,suggested: Noneanti-rabbit …SciScore for 10.1101/2021.04.19.440086: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization Number of cells highly infected (strong N staining) was counted in 12 random fields (magnification 20x) and % of cells expressing stress granule (G3BP1) signals in these were calculated. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Virus was detected using primary monoclonal rabbit antibodies directed against SARS-CoV-2 nucleocapsid (Sino Biological Inc., 40143-R001), and secondary anti-rabbit HRP conjugated antibodies (1:2000, Thermo Fisher Scientific). SARS-CoV-2 nucleocapsid ( Sino Biological Inc . , 40143-R001) ,suggested: Noneanti-rabbit HRPsuggested: NoneAntibodies: The following antibodies were used at the indicated dilutions: c-Myc (1:1000, Santa Cruz Biotechnology, sc-40), rabbit anti-G3BP1 (WB; 1:1000 c-Mycsuggested: (Santa Cruz Biotechnology Cat# sc-40 AC, RRID:AB_2857941)WBsuggested: Nonemouse APC-conjugated antibody directed against dsRNA J2 (IF 1:200, Scicons, 10010500) mouse anti-3xFlag M2 (WB 1:25000, Sigma, anti-3xFlagsuggested: NoneF1804), rabbit anti-Tubulin (WB 1:4000, Abcam, ab6046) anti-Tubulinsuggested: Nonedonkey anti-rabbit IgG (Invitrogen), goat anti-rabbit HRP conjugated antibody (WB 1:2000 or 1:5000, Thermo Fisher Scientific, 31460) donkey anti-rabbit IgGsuggested: Noneanti-rabbit IgGsuggested: (Thermo Fisher Scientific Cat# 31460, RRID:AB_228341)goat anti-mouse HRP conjugated antibody (WB 1:5000, Thermo Fisher Scientific, 31430) goat anti-mouse HRPsuggested: (Thermo Fisher Scientific Cat# 31430, RRID:AB_228307)anti-mouse HRPsuggested: (Thermo Fisher Scientific Cat# 31430, RRID:AB_228307)Thereafter, cells are incubated with primary antibodies against SARS-CoV-2 nucleocapsid ((1:500) Sino Biological Inc., 40143-R001) and G3BP1 ((1:500 SARS-CoV-2 nucleocapsidsuggested: NoneG3BP1suggested: None) Abcam, ab56574) followed by incubation with conjugated secondary antibodies anti-rabbit Alexa555 and anti-mouse Alexa488 (1:500, Thermo Fisher Scientific). anti-rabbitsuggested: Noneanti-mousesuggested: (Bioss Cat# bsm-4579M-Alexa488, RRID:AB_11071160)Proteins were separated with SDS-PAGE and Western blot analysis was performed using antibodies against SARS CoV-2 nucleocapsid, 3xFlag M2 (Sigma, F1804) and tubulin (Abcam, ab6046) antibodies against SARS CoV-2 nucleocapsid ,suggested: NoneF1804suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)tubulin ( Abcam ,suggested: (Abcam Cat# ab106042, RRID:AB_10898490)Experimental Models: Cell Lines Sentences Resources Cell culture, virus and reagents: HeLa and HEK293 cells were maintained in DMEM GlutaMAX containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FCS (all from Thermo Fisher Scientific) HEK293suggested: NoneLentivirus production, transductions and virus infection: Lenti-X 293T cells (Takara bio) grown in a 100 mm plate format were co-transfected with 4.5 μg psPAX2 (Didier Trono lab, Addgene plasmid #12260), 500 ng pMD2. 293Tsuggested: NoneFor transductions VeroE6 or HEK cells were seeded into greiner CELLSTAR® 96-well plates or 6-well plates (VWR) containing lentivirus in DMEM containing 2 % FBS and 1 μg/mL polybrene, and incubated for 72 h. HEKsuggested: NoneImmunoprecipitation of SARS-CoV/SARS-CoV2/MERS-CoV N proteins: Two 15 cm3 dishes were seeded with HeLa cells at 20% confluency. HeLasuggested: NoneVeroE6 cells were seeded in 8-well chamber slides (Sarstedts) and infected with SARS CoV-2 for 6 h. VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Envelope 3xFlag (pGBW-m4252867 was a gift from Ginkgo Bioworks & Benjie Chen (Addgene plasmid # 153626; http://n2t.net/addgene:153626; RRID: Addgene_153626) and pcDNA5/FRT/TO Myc SARS CoV2 N and N 2A was transfected into HEK293T cells using GeneJuice (Novagen, Darmstadt, Germany) following the manufacturer’s protocol (12 μg of DNA/sample in total). HEK293Tsuggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)Recombinant DNA Sentences Resources To generate the YFP-G3BPi inhibitor, the double FGDF motif from Semliki Forest virus nsP3 (RTTFRNKLPFTFGDFDEHEVDALASGITFGDFDDVL) or a control inhibitor (RTTFRNKLPATAGDFDEHEVDALASGITAGDADDVL) were fused to the C terminus of YFP by cloning the DNA encoding this into the pcDNA5/FRT/TO vector (Invitrogen). pcDNA5/FRT/TOsuggested: RRID:Addgene_19445)Lentivirus production, transductions and virus infection: Lenti-X 293T cells (Takara bio) grown in a 100 mm plate format were co-transfected with 4.5 μg psPAX2 (Didier Trono lab, Addgene plasmid #12260), 500 ng pMD2. psPAX2suggested: RRID:Addgene_12260)pMD2suggested: NoneTo generate transfer plasmids, four copies of inhibitory peptide (three copies for the Semilikiforest peptide) or control peptides with the binding motifs mutated were fused to C terminus of EGFP and cloned to pLJM1-EGFP vector (David Sabatini lab, Addgene plasmid #19319). pLJM1-EGFPsuggested: RRID:Addgene_19319)Assembly assay: pcDNA3.1 expression plasmids coding for the structural proteins of SARS CoV-2, Spike (S D614), Membrane (M) (was a gift from Jeremy Luban pcDNA3.1suggested: RRID:Addgene_79663)Envelope 3xFlag (pGBW-m4252867 was a gift from Ginkgo Bioworks & Benjie Chen (Addgene plasmid # 153626; http://n2t.net/addgene:153626; RRID: Addgene_153626) and pcDNA5/FRT/TO Myc SARS CoV2 N and N 2A was transfected into HEK293T cells using GeneJuice (Novagen, Darmstadt, Germany) following the manufacturer’s protocol (12 μg of DNA/sample in total). pGBW-m4252867suggested: NoneSoftware and Algorithms Sentences Resources The sample was analyzed using Illumina MiSeq v3 run, 1×150bp read setup, 20% PhiX by the NGS-NGI SciLifeLab facility. MiSeqsuggested: (A5-miseq, RRID:SCR_012148)Results were processed using in-house custom Python scripts described previsouly22. Pythonsuggested: (IPython, RRID:SCR_001658)To assess the quality of RiboVD library we analyzed the coverage percentage of the phage library over the library design using the NGS results of non-challenged naive library phage aliquotes. 95.50% of the peptide sequences designed to be in the library were confirmed by the NGS analysis of the naïve phage library. NGSsuggested: (PM4NGS, RRID:SCR_019164)The peptide data was combined with available information on SG localized proteins from mass spectroscopy of purified mammalian stress granules27, 52, 59 and from other studies based on the information listed in the HIPPIE database. HIPPIEsuggested: (HIPPIE, RRID:SCR_014651)Peptides were from GeneCust (France) with a purity of over 95%. GeneCustsuggested: NoneBioinformatic analysis: Proteomics data analysis was performed with Perseus65 and within the R environment (https://www.r-project.org/). https://www.r-project.org/suggested: (R Project for Statistical Computing, RRID:SCR_001905)MaxQuant output tables were filtered for ‘Reverse’, ‘Only identified by site modification’, and ‘Potential contaminants’ before data analysis. MaxQuantsuggested: (MaxQuant, RRID:SCR_014485)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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- No protocol registration statement was detected.
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