The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression
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SciScore for 10.1101/2021.05.28.446204: (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 not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The following antibodies were used for western blotting: mouse anti-GFP (1:5000; Clontech 632381), rabbit anti-Vinculin (1:1000, Abcam GR268234-50), nd mouse antiFLAG M2 (1:1000, Sigma-Aldrich SLBT7654), rabbit anti-RPS2 (1:2000, Bethyl labs A303-794A-M), rabbit anti-RPS3 (1:500, Proteintech 11990-1-AP), rabbit anti-RPS24 (1:1000, Bethyl labs A303-842A-T), rabbit anti-RACK1 (1:1000, Bethyl labs A302-545A), HRP goat anti-mouse IgG (1:10,000 SouthernBiotech 1031-05) and HRP goat … SciScore for 10.1101/2021.05.28.446204: (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 not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The following antibodies were used for western blotting: mouse anti-GFP (1:5000; Clontech 632381), rabbit anti-Vinculin (1:1000, Abcam GR268234-50), nd mouse antiFLAG M2 (1:1000, Sigma-Aldrich SLBT7654), rabbit anti-RPS2 (1:2000, Bethyl labs A303-794A-M), rabbit anti-RPS3 (1:500, Proteintech 11990-1-AP), rabbit anti-RPS24 (1:1000, Bethyl labs A303-842A-T), rabbit anti-RACK1 (1:1000, Bethyl labs A302-545A), HRP goat anti-mouse IgG (1:10,000 SouthernBiotech 1031-05) and HRP goat anti-rabbit IgG (1:10,000 SouthernBiotech 4030-05). anti-GFPsuggested: Noneanti-Vinculinsuggested: NoneantiFLAGsuggested: Noneanti-RPS2suggested: (Thermo Fisher Scientific Cat# A303-794A-M, RRID:AB_2781471)anti-RPS3suggested: (Proteintech Cat# 11990-1-AP, RRID:AB_2180758)anti-RPS24suggested: (Thermo Fisher Scientific Cat# A303-842A-M, RRID:AB_2781505)anti-RACK1suggested: (Antibodies-Online Cat# ABIN223360, RRID:AB_10849103)anti-mouse IgGsuggested: Noneanti-rabbit IgGsuggested: (SouthernBiotech Cat# 4030-05, RRID:AB_2687483)Experimental Models: Cell Lines Sentences Resources Cell culture and transfections: HEK293T cells were grown in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (Peak Serum) at 37°C and 5% CO2. HEK293Tsuggested: NoneRibosome purification: HeLa cell extract was prepared as described previously52. HeLasuggested: NoneRecombinant DNA Sentences Resources Full-length nsp1 was synthesized as a gene fragment from Integrated DNA technologies (IDT) and cloned into the EcoR1 restriction site of pGEX-6P-2 (GE healthcare) using in-fusion cloning (TakaraBio). pGEX-6P-2suggested: RRID:Addgene_131146)A T7 promoter upstream of the human β-globin 5’ UTR or the SARS-CoV-2 leader fused to a nano luciferase (HBB-nLuc and CoV-2 leader-nLuc, respectively) was synthesized by Twist Bioscience then subcloned by in-fusion cloning into an EcoRI site in a pUC57 destination vector. pUC57suggested: RRID:Addgene_40306)CoV-2 nsp1 was subcloned into the NotI site of pCDNA4-3X-FLAG-Halo using in-fusion cloning. pCDNA4-3X-FLAG-Halosuggested: NoneTruncation mutants Δ118-180 and Δ1-117 were PCR amplified from the parental vector pCDNA4-3x-FLAG-Halo-nsp1 and cloned as described above. pCDNA4-3x-FLAG-Halo-nsp1suggested: NoneThe CoV-2-leader nLuc sequence was synthesized by IDT and similarly cloned into the pLJM1 vector. pLJM1suggested: RRID:Addgene_73031)For the GFP cotransfection experiments, 1×106 cells were seeded into each well of a 6-well plate and transfected the next day with 100 ng of pCDNA-GFP and 900 ng of the indicated pCDNA4-3X-FLAG-Halo-nsp1 construct. pCDNA-GFPsuggested: RRID:Addgene_160697)For the luciferase assays, 4.5×104 cells were seeded into each well of a 96-well plate and transfected the next day with 10 ng of HBB-nLuc or CoV2L-nLuc and 25 ng of the indicated C-terminally 3xFLAG tagged nsp1 (pCDNA4-nsp1-3xFLAG) construct. pCDNA4-nsp1-3xFLAGsuggested: NoneSoftware and Algorithms Sentences Resources Transfections were carried out using PolyJet™ (SignaGen labs) according to the manufacturer’s DNA transfection protocol and cells were harvested 24 hr post transfection. PolyJet™suggested: NoneFor immunoprecipitations, cell pellets were lysed in 1 mL of lysis buffer (Buffer A) containing 20mM Tris-KOH pH 7.5, 150mM KOAc, 5mM MgCl2, 5% glycerol, 1X HaltTM protease inhibitor cocktail (ThermoFisher), 1mM DTT (Danville Scientific) and 0.5% NP-40 (Danville Scientific) and placed on a rotating wheel for 30 min. ThermoFishersuggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
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.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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