Nsp1 proteins of human coronaviruses HCoV-OC43 and SARS-CoV2 inhibit stress granule formation

Article activity feed

1. Version published to 10.1371/journal.ppat.1011041

   Dec 19, 2022
2. 

   ScreenIT

   May 5, 2022

   SciScore for 10.1101/2022.05.02.490272: (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	Quantification of SG-positive cells was performed by counting the number of cells with at least two discrete cytoplasmic foci from at least 3 randomly selected fields of view, analysing >100 cells per treatment in each replicate.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After 1-h blocking with 5% bovine serum albumin (BSA, BioShop, Burlington, ON, Canada) in PBS, staining was performed overnight at +4°C with antibodies to the following targets: CoV2 N (1:400; rabbit, Novus Biologicals, NBP3-05730); eIF3B (1:400; rabbit, Bethyl Labs, …

   SciScore for 10.1101/2022.05.02.490272: (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	Quantification of SG-positive cells was performed by counting the number of cells with at least two discrete cytoplasmic foci from at least 3 randomly selected fields of view, analysing >100 cells per treatment in each replicate.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After 1-h blocking with 5% bovine serum albumin (BSA, BioShop, Burlington, ON, Canada) in PBS, staining was performed overnight at +4°C with antibodies to the following targets: CoV2 N (1:400; rabbit, Novus Biologicals, NBP3-05730); eIF3B (1:400; rabbit, Bethyl Labs, A301761A); eIF4G (1:200; rabbit, Cell Signaling, #2498); G3BP1 (1:400; mouse, BD Transduction, 611126); G3BP2 (1:1000; rabbit, Millipore Sigma,	NBP3-05730); eIF3B suggested: None
   Alexa Fluor (AF)-conjugated secondary antibodies used were: donkey anti-mouse IgG AF488 (Invitrogen, A21202)	anti-mouse IgG suggested: (Molecular Probes Cat# A-21202, RRID:AB_141607)
   Aliquots of lysates thawed on ice were incubated at 95°C for 3 min, cooled on ice, separated using denaturing PAGE, transferred onto PVDF membranes using Trans Blot Turbo Transfer System with RTA Transfer Packs (Bio-Rad Laboratories, Hercules, CA, USA) according to manufacturer’s protocol and analysed by immunoblotting using antibody-specific protocols.	antibody-specific protocols . suggested: None
   Antibodies to the following targets were used: β-actin (1:2000; HRP-conjugated, mouse, Santa Cruz Biotechnology, sc- 47778); CoV2 N (1:1,000; rabbit, Novus Biologicals,	β-actin suggested: (Santa Cruz Biotechnology Cat# sc-47778 HRP, RRID:AB_2714189)
   Puromycin incorporation into nascent polypeptides was visualised using anti-puromycin antibody (1:6,000; mouse, MilliporeSigma, MABE343).	anti-puromycin suggested: (Millipore Cat# MABE343, RRID:AB_2566826)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cells: Human Embryonic Kidney (HEK) 293A cells and human colon adenocarcinoma (HCT-8) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with heat-inactivated 10% fetal bovine serum (FBS), and 2 mM L-glutamine (all purchased from Thermo Fisher Scientific (	HEK suggested: None HCT-8 suggested: None
   BEAS-2B cells were cultured in Bronchial Epithelial Cell Growth Medium (BEGM, Lonza, Kingston, ON, Canada) on plates prepared with coating media (0.01 mg/ml fibronectin, 0.03 mg/mL bovine collagen type I, and 0.01 mg/ml bovine serum albumin (all from Millipore Sigma, Oakville, ON, Canada) dissolved in Basal Epithelial Cell Growth Medium (BEBM, Lonza)). 293A and BEAS-2B cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA)	BEAS-2B suggested: None
   For SARS-CoV-2 stocks, Vero E6 cells in a confluent T-175 cm2 flask were infected at a MOI of 0.01 for 1 h at 37°C in 2.5 mL of serum-free DMEM with intermittent shaking every 10 min.	Vero E6 suggested: None
   To generate lentivirus stocks, HEK 293T cells (ATCC) were reverse- transfected with polyethylenimine (PEI, Polysciences, Warrington, PA, USA) and the following plasmids for lentiviral generation: pLJM1-B*	HEK 293T suggested: None
   Transfection: 293A cells were seeded into 20-mm wells of 12-well cluster dishes with or without glass coverslips and the next day transfected with 500 ng DNA mixes/well containing expression vectors (250 ng) and pUC19 filler DNA (250 ng) using Fugene HD (Promega, Madison, WI, USA) according to manufacturer’s protocol.	293A suggested: None
   Recombinant DNA
   Sentences	Resources
   Then, coding sequences were inserted between EcoRI and XhoI sites into pCR3.1-EGFP vector (75) to generate pCR3.1-EGFP-OC43-N, pCR3.1-EGFP-CoV2-N, pCR3.1-EGFP-OC43-Nsp1, pCR3.1-EGFP-CoV2-Nsp1, and pCR3.1-EGFP-OC43-Nsp15 plasmids.	pCR3.1-EGFP suggested: None pCR3.1-EGFP-OC43-N suggested: None pCR3.1-EGFP-CoV2-N suggested: None pCR3.1-EGFP-OC43-Nsp1 suggested: None pCR3.1-EGFP-CoV2-Nsp1 suggested: None pCR3.1-EGFP-OC43-Nsp15 suggested: None
   To generate N-terminally HA-tagged Nsp1 constructs, coding sequences were inserted between KpnI and XhoI sites into 3xHA-miniTurbo-NLS_pCDNA3 vector (a gift from Alice Ting, Addgene plasmid # 107172) to generate pCDNA3-HA-OC43-Nsp1 and pCDNA3-HA-CoV2-Nsp1 vectors (miniTurbo-NLS coding sequence was replaced by Nsp1 sequences)	3xHA-miniTurbo-NLS_pCDNA3 suggested: RRID:Addgene_107172) pCDNA3-HA-OC43-Nsp1 suggested: None pCDNA3-HA-CoV2-Nsp1 suggested: None
   Amino acid substitutions in pCDNA3-HA-CoV2-Nsp1 vector were introduced using Phusion PCR mutagenesis (New England Biolabs) to generate pCDNA-HA-CoV2-Nsp1(R99A) and pCDNA-HA-CoV2-Nsp1(R124A,K125A) vectors.	pCDNA-HA-CoV2-Nsp1 suggested: None
   To generate lentivirus vectors pLJM1-ACE2-BSD, pLJM1-EGFP-BSD, and pLJM1-EGFP- G3BP1-BSD, the PCR-amplified ACE2, EGFP, and G3BP1 coding sequences were inserted into the multicloning site of pLJM1-B* vector (76).	pLJM1-EGFP-BSD suggested: None pLJM1-EGFP- G3BP1-BSD suggested: None pLJM1-B* suggested: None
   backbone-based constructs, pMD2.G, and psPAX2. pMD2.	pMD2 . G suggested: None psPAX2 suggested: RRID:Addgene_12260) pMD2 suggested: None
   Generation of stably transduced cell lines: To generate 293A-ACE2 cells, 293A cells were stably transduced with a lentivirus vector encoding ACE2 (pLJM1-ACE2-BSD) and selected and maintained in 10 μg/mL Blasticidin S HCl (Thermo Fisher).	pLJM1-ACE2-BSD suggested: None
   Transfection: 293A cells were seeded into 20-mm wells of 12-well cluster dishes with or without glass coverslips and the next day transfected with 500 ng DNA mixes/well containing expression vectors (250 ng) and pUC19 filler DNA (250 ng) using Fugene HD (Promega, Madison, WI, USA) according to manufacturer’s protocol.	pUC19 suggested: RRID:Addgene_50005)
   Where indicated, the amount of filler DNA was reduced to 150 ng and 100 ng of the pCR3.1- EGFP plasmid was co-transfected with expression vectors for Nsp1 proteins.	pCR3.1- suggested: None
   Software and Algorithms
   Sentences	Resources
   Analysis of SG number and size was performed on cropped images of individual cells using ImageJ software Analyze Particles function after automatic background substraction and thresholding.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Aliquots of lysates thawed on ice were incubated at 95°C for 3 min, cooled on ice, separated using denaturing PAGE, transferred onto PVDF membranes using Trans Blot Turbo Transfer System with RTA Transfer Packs (Bio-Rad Laboratories, Hercules, CA, USA) according to manufacturer’s protocol and analysed by immunoblotting using antibody-specific protocols.	Bio-Rad Laboratories suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
   For analyses of protein band intensities, western blot signals were quantified using Bio-Rad Image Lab 5.2.1 software and values normalized to the Stain-free signal for each lane.	Bio-Rad Image suggested: None
   Statistical analyses for each data set are described in figure legends and were performed using GraphPad Prism 8 software.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

   ---

   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.

   ---

   Read the original source
3. 

   Version published to 10.1101/2022.05.02.490272v1 on bioRxiv

   May 2, 2022