Parsing the role of NSP1 in SARS-CoV-2 infection

Version published to 10.1016/j.celrep.2022.110954

Jun 1, 2022

SciScore for 10.1101/2022.03.14.484208: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	Field Sample Permit: Handling and working with SARS-CoV-2 were conducted in a BSL3 facility in accordance with the biosafety guidelines of the Israel Institute for Biological Research (IIBR), or in a BSL3 facility at The University of Texas Medical Branch (UTMB). Euthanasia Agents: Golden Syrian hamsters (females; 4-5 weeks old) were intranasally infected with 1000 pfu of virus in 50 μl of inoculum while under isoflurane anesthesia.
Sex as a biological variable	not detected.
Randomization	A DIG-labeled random-primed probe, corresponding to nucleotides 28,999 to 29,573 of the SARS-CoV-2 genome (Kamitani et al., 2006; Xie et al., 2020), was used to detect SARS-CoV-2 mRNAs and visualized by DIG luminescent detection kit (Roche, Indianapolis, IN), according to the manufacturer’s protocol.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	Authentication: Calu3 and 293T cells were authenticated by ATCC using STR profiling. Contamination: All cell lines tested negative for mycoplasma.

Table 2: Resources

Antibodies
Sentences	Resources
Briefly, paraffin-embedded sections were firstly subjected to immunostaining with a commercially available and diluted (1:5,000) rabbit-anti-SARS-CoV-2 spike (S) protein (ab272504, Abcam Plc., Cambridge, UK), followed by staining with a peroxidase-conjugated secondary antibody.	rabbit-anti-SARS-CoV-2 spike ( S ) protein ( ab272504 suggested: None
For the detection of N protein, an affinity-purified rabbit anti-SARS-CoV N polyclonal antibody (Harcourt et al., 2020) was used as the primary antibody, followed by Alexa Fluor 488-conjugated secondary antibody (Invitrogen) and DAPI counterstaining, using SlowFade Gold antifade reagent (Invitrogen).	anti-SARS-CoV suggested: None
For the detection of nsp1 protein, the anti-SARS-CoV-1 nsp1 antibody was used as the primary antibody, followed by Alexa Fluor 594-conjugated secondary antibody (Invitrogen) and DAPI counterstaining.	anti-SARS-CoV-1 nsp1 suggested: None
Cells were washed and labeled with anti-rabbit FITC conjugated antibody at a 1:200 dilution and with DAPI (4′,6-diamidino-2-phenylindole) at a 1:200 dilution.	anti-rabbit FITC suggested: (Bioss Cat# bs-0708R-FITC, RRID:AB_11075028)
Secondary antibodies used were Goat anti-rabbit or Goat anti-mouse (IRDye 800CW or IRDye 680RD, Li-Cor).	anti-rabbit suggested: None anti-mouse suggested: None
An affinity-purified rabbit anti-SARS-CoV-1 nsp1 polyclonal antibody (Narayanan et al., 2015) was used as the primary antibody, and an HRP-linked anti-rabbit IgG (Cell Signaling Technology) was used as the secondary antibody.	anti-SARS-CoV-1 suggested: None anti-rabbit IgG ( Cell Signaling Technology ) suggested: None
Experimental Models: Cell Lines
Sentences	Resources
African green monkey kidney clone E6 cells (Vero E6, ATCC® CRL-1586™) were grown in growth medium DMEM containing 10% FBS, MEM NEAA, 2 mM L-glutamine, 100 Units/ml penicillin, 0.1 mg/ml streptomycin, 12.5 Units/ml nystatin (P/S/N), all from Biological Industries, Israel]	Vero E6 suggested: None
Calu3 and 293T cells were authenticated by ATCC using STR profiling.	293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
Viral titers: For determination of growth rate of SARS-CoV-2 WT or SARS-CoV-2 mut viruses, Vero E6 and Calu3 cells were seeded in 12-well plates (5E5 and 1E6 cells/well respectively).	Calu3 suggested: RRID:CVCL_EQ19)
Virus titers in the tissues were determined on confluent Vero E6/TMPRSS2 cells obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (1819).	Vero E6/TMPRSS2 suggested: None
Recombinant DNA
Sentences	Resources
SARS-CoV-2 nsp1 ORF, carrying a C-terminal myc-tag, was cloned into pCAGGS-MCS, resulting in pCAGGS-nsp1-WT.	pCAGGS-MCS suggested: None pCAGGS-nsp1-WT suggested: None
Similarly, SARS-CoV-2 nsp1 carrying a deletion of the amino acids 155 to 165, and SARS-CoV-2 nsp1 carrying R124A and K125A mutations, were cloned into pCAGGS to generate pCAGGS-nsp1-ΔRB and pCAGGS-nsp1-CD, respectively.	pCAGGS suggested: RRID:Addgene_127347) pCAGGS-nsp1-ΔRB suggested: None pCAGGS-nsp1-CD suggested: None
Luciferase assay: Vero E6 cells in 24-well plates were co-transfected, in triplicate, with pRL-EMCV-FL reporter plasmid and the indicated pCAGGS-based nsp1 expression plasmids using TransIT-LT1 reagent (Mirus).	pRL-EMCV-FL suggested: None
Plasmids and cloning: pLVX-EF1alpha-SARS-CoV-2-nsp1-2XStrep-IRES-Puro (nsp1-WT) was kindly provided by N. Krogan.	pLVX-EF1alpha-SARS-CoV-2-nsp1-2XStrep-IRES-Puro suggested: RRID:Addgene_141367)
The mutations to generate nsp1-ΔRB and nsp1-CD were introduced by amplifying the nsp1-WT plasmid so that it became a linear fragment containing the desired mutations (primers 1-4 listed in Supplementary Table 1).	nsp1-WT suggested: None
Software and Algorithms
Sentences	Resources
The images for SARS-CoV-2 N protein were collected using an Olympus BX53 microscope with a 40X objective lens at UTMB and processed with ImageJ (NIH) software.	UTMB suggested: None
Alignment was performed using Bowtie v.	Bowtie suggested: (Bowtie, RRID:SCR_005476)
Differential expression analysis was done using DESeq2 (Love et al., 2014)	DESeq2 suggested: (DESeq, RRID:SCR_000154)
Output.bam files from STAR were used as input for the GRAND-SLAM analysis(Jürges et al., 2018) with default parameters and with trimming of 5 nucleotides in the 5′ and 3′ends of each read.	STAR suggested: (STAR, RRID:SCR_004463)
Reactive bands were detected by Odyssey CLx infrared imaging system (Li-Cor) and quantified using Fiji (ImageJ) as detailed in https://imagej.nih.gov/nih-image/manual/tech.html.	Fiji suggested: (Fiji, RRID:SCR_002285) ImageJ suggested: (ImageJ, RRID:SCR_003070)
Graphics: Figure 1a and extended figure 2c were created using BioRender.	BioRender suggested: (Biorender, RRID:SCR_018361)
Fluorescence-activated cell sorting figures were created with FlowJo and the rest of the figures were drawn with ggplot2 in R.	FlowJo suggested: (FlowJo, RRID:SCR_008520) ggplot2 suggested: (ggplot2, RRID:SCR_014601)

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: We detected the following sentences addressing limitations in the study:

However, a major limitation of these measurements is that viral mRNA replication and transcription is massive, and occurs within a unique endomembrane system (Wolff et al., 2020) that would thus render much of the viral mRNA inaccessible to ribosomes. This inaccessibility of potentially large portions of viral RNA will skew our measurements and it is likely the true translation efficiency of viral transcripts is significantly higher. Comparing the translation efficiencies of viral and cellular transcripts in cells infected with CoV2-wt or with CoV2-mut allowed us to overcome this limitation and to demonstrate that in infected cells nsp1 indeed specifically promotes the translation of viral transcripts compared to cellular transcripts, as we observed that in cells infected with CoV2-mut, viral genes are translated even less efficiently compared to their cellular counterparts. However, at the same time our measurements of the overall translation capacity in infected cells, using incorporation of OPP into nascent chains, show that nsp1-WT leads to a major reduction in the absolute levels of translation in infected cells. Since the vast majority of mRNA in infected cells is viral mRNA this means that viral mRNAs translation is likely also inhibited in the presence of nsp1. Together these results support a model in which nsp1 acts as a strong inhibitor of translation that tightly binds to ribosomes and thus inhibits the translation of both viral and cellular mRNAs. Viral transcrip...

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:

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No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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Parsing the role of NSP1 in SARS-CoV-2 infection

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