SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation

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   ScreenIT

   Jan 23, 2022

   SciScore for 10.1101/2022.01.19.476693: (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
   Antibodies, siRNAs, shRNAs, and plasmids: The following antibodies were used: rabbit anti-eIF4E2 (Genetex, GTX103977 and GTX64395), rabbit anti-GIGYF1 (Bethyl Laboratories, A304-132A), rabbit anti-GIGYF2 (Bethyl Laboratories, A303-732A), sheep anti-SARS-CoV-2-NSP2 (MRC PPU reagents and Services, DA105), mouse anti-β-actin (Sigma, A5441), rabbit anti-STAT1 (Cell Signaling, 14994), rabbit anti-phospho-STAT1 (Tyr701; Cell signaling, 7649), rabbit anti-TLR3 (Cell signaling, 6961), rabbit …

   SciScore for 10.1101/2022.01.19.476693: (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
   Antibodies, siRNAs, shRNAs, and plasmids: The following antibodies were used: rabbit anti-eIF4E2 (Genetex, GTX103977 and GTX64395), rabbit anti-GIGYF1 (Bethyl Laboratories, A304-132A), rabbit anti-GIGYF2 (Bethyl Laboratories, A303-732A), sheep anti-SARS-CoV-2-NSP2 (MRC PPU reagents and Services, DA105), mouse anti-β-actin (Sigma, A5441), rabbit anti-STAT1 (Cell Signaling, 14994), rabbit anti-phospho-STAT1 (Tyr701; Cell signaling, 7649), rabbit anti-TLR3 (Cell signaling, 6961), rabbit anti-v5 (abcam, ab9116), mouse anti-FLAG (abcam, ab49763).	anti-eIF4E2 suggested: (GeneTex Cat# GTX103977, RRID:AB_2036842) GTX64395 suggested: None anti-GIGYF1 suggested: (Bethyl Cat# A304-132A, RRID:AB_2621381) anti-GIGYF2 suggested: (Thermo Fisher Scientific Cat# A303-732A, RRID:AB_11205815) anti-SARS-CoV-2-NSP2 suggested: None anti-β-actin suggested: (Sigma-Aldrich Cat# A5441, RRID:AB_476744) anti-STAT1 suggested: (Cell Signaling Technology Cat# 14994, RRID:AB_2737027) anti-phospho-STAT1 suggested: (Cell Signaling Technology Cat# 7649, RRID:AB_10950970) anti-TLR3 suggested: None anti-v5 suggested: (Abcam Cat# ab9116, RRID:AB_307024) anti-FLAG suggested: (Abcam Cat# ab49763, RRID:AB_869428)
   Experimental Models: Cell Lines
   Sentences	Resources
   A549 (ATCC), were cultured in RPMI, also supplemented with 10% FBS and 1% P/S. Calu-3 (ATCC) were cultured in EMEM (Eagle Minimal Essential Medium) supplemented with 20% FBS and 1% P/S. WT, 4EHP-knockout and GIGYF2-knockout HEK293 cells were maintained in DMEM supplemented with 10% FBS, 1% P/S, 100 µg/mL zeocin (Thermo Fisher Scientific, R25001), and 15 µg/mL blasticidin (Thermo Fisher Scientific, R210-01)17.	A549 suggested: RRID:CVCL_4Z15) Calu-3 suggested: None
   To generate gene knockout Flp-In T-REx HEK293 cells, 130,000 cells were transfected with the corresponding guide sequence containing pSpCas9(BB)-2A-GFP plasmid.	Flp-In T-REx suggested: RRID:CVCL_6E37)
   Lentivirus production: Lentivirus pseudovirions were produced by transfecting HEK293T cells using Lipofectamine 2000 and 10 μg shRNAs, lenti-Cas9-Blast, pLenti-CRISPRv2 inserted with CRISPR guide RNA (gRNA) for gene knockout plasmids, or Lenti-ORF plasmid, 6 μg psPAX2 (Addgene, plasmid 12260) and 4 μg pMD2.G (Addgene, plasmid 12259).	HEK293T suggested: None
   Briefly, the IFN-β–Luc was co-transfected with pRL-TK in wild type and 4EHP-KO HEK293 cells using Lipofectamine 2000 according to the manufacturer′s protocol (Invitrogen).	HEK293 suggested: None
   Recombinant DNA
   Sentences	Resources
   The pLenti-CMV-GFP-Puro (Addgene, 17448), pLenti-CMV-Luc-Puro (Addgene, 17477), Lenti-4EHP (Sigma, TRCN0000474313), and pLenti-X2-Zeo-DEST (Addgene, 21562) were used to generate cells which stably express GFP, luciferase,4EHP, and NSP2, respectively.	pLenti-CMV-GFP-Puro suggested: None pLenti-CMV-Luc-Puro suggested: None
   The Lenti-Cas9-Blast (Addgene, plasmid 52962) and pLenti-CRISPRv2 (Addgene, plasmid 52961) were used to generate the knockout.	pLenti-CRISPRv2 suggested: None
   Briefly, the forward and reverse strand oligodeoxynucleotides were annealed and ligated into pSpCas9(BB)-2A-GFP (Addgene, PX458, Plasmid #48138) linearized with BbsI (Thermo Fisher Scientific, ER1011).	pSpCas9(BB)-2A-GFP suggested: RRID:Addgene_48138)
   After transformation, the guide sequence containing pSpCas9(BB)2A-GFP plasmids were isolated and sequence-verified.	BB)2A-GFP suggested: None
   To generate gene knockout Flp-In T-REx HEK293 cells, 130,000 cells were transfected with the corresponding guide sequence containing pSpCas9(BB)-2A-GFP plasmid.	BB)-2A-GFP suggested: RRID:Addgene_104437)
   Lentivirus production: Lentivirus pseudovirions were produced by transfecting HEK293T cells using Lipofectamine 2000 and 10 μg shRNAs, lenti-Cas9-Blast, pLenti-CRISPRv2 inserted with CRISPR guide RNA (gRNA) for gene knockout plasmids, or Lenti-ORF plasmid, 6 μg psPAX2 (Addgene, plasmid 12260) and 4 μg pMD2.G (Addgene, plasmid 12259).	Lenti-ORF suggested: None psPAX2 suggested: RRID:Addgene_12260) pMD2.G suggested: RRID:Addgene_12259)
   Plasmid construction: Synthesized viral coding sequences were incorporated into Gateway-compatible Entry vectors; pDONR207 SARS-CoV-2 NSP1 (Addgene, 141255), pDONR223 SARS-CoV-2 NSP2 (Addgene, 141256) and expression clones with N-terminal fusion tags were produced simply by Gateway cloning (Gateway™ LR Clonase™ II Enzyme mix, Invitrogen, 11791020).	pDONR207 suggested: RRID:Addgene_100600) pDONR223 suggested: RRID:Addgene_60532)
   The Lenti-NSP2 was constructed by using the destination vector, pLenti-X2-Zeo-DEST (749-3) [a gift from Eric Campeau (Addgene, 21562)] and the donor vector, pDONR223 SARS-CoV-2 NSP2.	pLenti-X2-Zeo-DEST suggested: None
   The Lenti-NSP2 plasmid was used to generate NSP2 stable cell lines by following the lentivirus production method section.	Lenti-NSP2 suggested: None
   Briefly, the IFN-β–Luc was co-transfected with pRL-TK in wild type and 4EHP-KO HEK293 cells using Lipofectamine 2000 according to the manufacturer′s protocol (Invitrogen).	pRL-TK suggested: RRID:Addgene_11313)
   The psiCHECK-2 control vector (Promega, C8021) and the psiCHECK-RL-Ifnb1 3′ UTR vector were described before26.	psiCHECK-2 suggested: RRID:Addgene_35672) psiCHECK-RL-Ifnb1 3′ UTR suggested: None
   Software and Algorithms
   Sentences	Resources
   Quantification and Statistical Analyses: Statistical tests were performed using Prism 6 (GraphPad).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   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).

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 24. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   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.

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

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   Version published to 10.1101/2022.01.19.476693v1 on bioRxiv

   Jan 20, 2022