SARS-CoV-2 ORF6 disrupts innate immune signalling by inhibiting cellular mRNA export

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1. Version published to 10.1371/journal.ppat.1010349

   Aug 25, 2022
2. 

   ScreenIT

   Feb 12, 2022

   SciScore for 10.1101/2022.02.08.479664: (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	Contamination: Stock cells were tested for mycoplasma contamination and authenticated by short-tandem repeat (STR) profiling. Authentication: Stock cells were tested for mycoplasma contamination and authenticated by short-tandem repeat (STR) profiling.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Primary antibodies used were: anti-HIV-1 p24 (ARP432, CFAR), anti-GFP (sc-9996, SCBT), anti-HA (C29F4, CST), anti-Strep-tag	Primary antibodies used were: anti-HIV-1 p24 ( ARP432 , CFAR) , anti-GFP ( sc-9996 , SCBT) , anti-HA ( C29F4 , CST) suggested: None

   SciScore for 10.1101/2022.02.08.479664: (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	Contamination: Stock cells were tested for mycoplasma contamination and authenticated by short-tandem repeat (STR) profiling. Authentication: Stock cells were tested for mycoplasma contamination and authenticated by short-tandem repeat (STR) profiling.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Primary antibodies used were: anti-HIV-1 p24 (ARP432, CFAR), anti-GFP (sc-9996, SCBT), anti-HA (C29F4, CST), anti-Strep-tag	Primary antibodies used were: anti-HIV-1 p24 ( ARP432 , CFAR) , anti-GFP ( sc-9996 , SCBT) , anti-HA ( C29F4 , CST) suggested: None anti-HIV-1 suggested: None anti-GFP suggested: (Santa Cruz Biotechnology Cat# sc-9996, RRID:AB_627695)
   Secondary antibodies include, anti-mouse (61-6520, Thermo Fisher)	anti-mouse suggested: (Innovative Research Cat# 61-6520, RRID:AB_138451)
   Cells were labelled with the following primary antibodies, anti-Nup98 or anti-ORF6 diluted in 4% BSA in PBS for 1 hour at RT.	anti-Nup98 suggested: None anti-ORF6 suggested: None
   Cells were then incubated with the following secondary antibodies, donkey anti-rabbit-AF568 (ab175692, Abcam) and donkey anti-sheep AF568 (A-21099, Invitrogen) diluted in 4% BSA in PBS for 1 hour at RT.	anti-rabbit-AF568 suggested: None donkey anti-sheep AF568 (A-21099, Invitrogen) diluted in 4% BSA in PBS for 1 hour at RT. suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines: HEK293T, HeLa, Mus dunni tail fibroblast (MDTF) and Vero cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher), supplemented with 10% heat-inactivated foetal bovine serum (FBS, Biosera) and 1% Penicillin/Streptomycin (Sigma).	HeLa suggested: None Vero suggested: None
   Immunoprecipitation: HEK293T cells were grown in 10 cm2 culture plates transfected and incubated for 16 hrs after which media was replaced.	HEK293T suggested: None
   Viruses were titrated by plaque assay on Vero E6 cells.	Vero E6 suggested: RRID:CVCL_XD71)
   Recombinant DNA
   Sentences	Resources
   The plasmids used to generate HIV-1 and MLV VLPs for transduction of ORFs, pCMVΔ8.91 and pHIT60 respectively, and pVSV-G have been described before [38, 39].	pCMVΔ8.91 suggested: None pVSV-G suggested: RRID:Addgene_138479)
   For the initial experiments on Gag expression (Figs 1A-C and S1A and B Figs) and ORF6 co-expression with Rae1 (Fig 2A), a pLVX-IRES-Puro vector encoding an individual ORF was used.	pLVX-IRES-Puro suggested: RRID:Addgene_140240)
   The pLVX-StrepII-ORF-IRES-Puro plasmids were then cloned using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB) from the pTriEX-6 plasmids, using the primers listed in Table 1.	pLVX-StrepII-ORF-IRES-Puro suggested: None pTriEX-6 suggested: None
   For the remaining assays that required ORF detection or immunoprecipitation, a pLVX-EF1α-SARS-CoV-2-ORF-2xStrep/2xStrep-ORF -IRES-Puro encoding one of the nine ORFs or pLVX-EF1α-eGFP-2xStrep-IRES-Puro plasmid was used, referred to as twin-strep-tag.	pLVX-EF1α-SARS-CoV-2-ORF-2xStrep/2xStrep-ORF -IRES-Puro suggested: None pLVX-EF1α-eGFP-2xStrep-IRES-Puro suggested: None
   These plasmids were kindly gifted from Nevan Krogan (Addgene plasmid #141383-4, #141387-90, #141392-95); http://n2t.net/addgene:141383-4, 141387-90, 141392-95; RRID:Addgene_141383-4, 141387-90, 141392-95) [5].	#141383-4 suggested: None
   To generate the pLVX-EF1α-CoV-1-ORF6-2xStrep-IRES-Puro plasmid, the ORF6(CoV-1)-2XStrep gene was synthesised by GeneArt and cloned in using the EcoRI and BamHI restriction sites.	pLVX-EF1α-CoV-1-ORF6-2xStrep-IRES-Puro suggested: None
   ORF6 mutations were introduced into the pLVX-StrepII-SARS-CoV-2-ORF6-IRES-Puro and pLVX-EF1α-SARS-CoV-2-ORF6-2xStrep-IRES-Puro plasmids using the QuickChange II-XL site-directed mutagenesis kit (Agilent) according to the manufacturer’s instructions using SDM primers listed below.	pLVX-StrepII-SARS-CoV-2-ORF6-IRES-Puro suggested: None pLVX-EF1α-SARS-CoV-2-ORF6-2xStrep-IRES-Puro suggested: None
   The following plasmids were sourced from Addgene, psfGFP-N1 (Addgene, #54737) and pmApple-N1 (Addgene, #54567).	pmApple-N1 suggested: None
   pCMVsport6Rae1 (MGC:117333) was purchased from Horizon Discovery Biosciences Limited.	pCMVsport6Rae1 suggested: None
   Virus-like particles (VLP) production: HIV-1 and MLV virus-like particles were generated by co-transfecting HEK293T cells with plasmids, pCMVΔ8.91 (HIV-1) or pHIT60 (MLV) and pVSV-G using lipofectamine 2000 (11668019, Invitrogen).	pHIT60 suggested: None
   For generating GFP reporter VLPs, pCSGW (HIV-1) or pczCFG2fEGFPf (MLV) were also added [39, 40].	pCSGW suggested: None
   Subcellular fractionation: HEK293T cells were transfected with psfGFP-N1 and either treated with LMB (Merck) at 5nM; or co-transfected with plasmids expressing pLVX-EF1α-ORF6, ORF6(M58A) or ORF9b [5].	psfGFP-N1 suggested: None pLVX-EF1α-ORF6 suggested: None
   Software and Algorithms
   Sentences	Resources
   The following primers and probes were used; sfGFP: for 5’-GCGCACCATCAGCTTCAAGG, rev 5’-GTGTCGCCCTCGAACTTCAC and probe 5’-FAM-CGGCACCTACAAGACCCGCGC-TAMRA. mRNAseq: The quality of extracted RNA was checked by Agilent TapeStation 42000 on Agilent RNA ScreenTape (Agilent technologies) before proceeding to library preparation.	Agilent TapeStation suggested: (Agilent TapeStation Laptop, RRID:SCR_019547)
   RNA-seq libraries were generated using the NEBNext Ultra Directional RNA Library Prep kit for Illumina with NEBNext® Poly(A) mRNA Magnetic Isolation Module (both New England BioLabs).	NEBNext® suggested: None Poly(A suggested: None
   Enriched GO terms (Biological process) were filtered and mapped using the REViGO tool available at http://revigo.irb.hr/ [41].	REViGO suggested: (REViGO, RRID:SCR_005825)
   Normalisation and differential expression analyses were conducted within DESeq2 [43]: normalisation factors for gene-level read counts were produced using counts for the D. melanogaster spike-in controls and differential gene expression calculations were subsequently performed for Human genes alone using the pre-calculated size factors.	DESeq2 suggested: (DESeq, RRID:SCR_000154)
   Statistics: Statistical analysis was performed using GraphPad Prism 9 software. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, ns – not significant).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: Thank you for sharing your data.

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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: We did not find any issues relating to colormaps.

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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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3. 

   Version published to 10.1101/2022.02.08.479664 on bioRxiv

   Feb 9, 2022