SARS-CoV-2 ORF6 Disrupts Bidirectional Nucleocytoplasmic Transport through Interactions with Rae1 and Nup98

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1. Version published to 10.1128/mbio.00065-21

   Apr 27, 2021
2. 

   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2020.08.03.234559: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Specimen collection and whole genome sequencing of SARS-CoV-2 positive clinical specimens: Whole genome sequencing of SARS-CoV-2 positive clinical specimens was conducted as part of an ongoing University of Washington Institutional Review Board-approved study (STUDY00000408) (35–38).
   Randomization	not detected.
   Blinding	mCherry fluorescence intensities were measured with ImageJ v1.53a by an individual blinded to experimental design.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Blotting was performed using the following primary antibodies: 1:1,000 anti-GFP …

   SciScore for 10.1101/2020.08.03.234559: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Specimen collection and whole genome sequencing of SARS-CoV-2 positive clinical specimens: Whole genome sequencing of SARS-CoV-2 positive clinical specimens was conducted as part of an ongoing University of Washington Institutional Review Board-approved study (STUDY00000408) (35–38).
   Randomization	not detected.
   Blinding	mCherry fluorescence intensities were measured with ImageJ v1.53a by an individual blinded to experimental design.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Blotting was performed using the following primary antibodies: 1:1,000 anti-GFP (Cell Signaling; clone 4B10), 1:500 anti-mCherry (	anti-GFP suggested: (Cell Signaling Technology Cat# 2955, RRID:AB_1196614)
   e IgG secondary antibody (Licor) or 1:5,000 IRDye 800CW anti-Rabbit IgG secondary antibody (Licor).	anti-Rabbit IgG suggested: None
   Western blotting using the elutes from affinity purification and the pre-purified input lysates were performed as described above with the following primary antibodies: 1:1,000 anti-GFP, 1:1,000 anti-alpha Tubulin, 1:2,000 anti-Rae1 (Abcam; clone EPR6923) and 1:1,000 anti-Nup98 (Abcam; clone 2H10).	anti-alpha Tubulin suggested: None anti-Rae1 suggested: None anti-Nup98 suggested: None
   Western blotting was performed as described above with the following primary antibodies: 1:1,000 anti-GFP, 1:500 anti-mCherry, 1:1,000 anti-alpha Tubulin, and 1:1,000 anti-FLAG (Sigma; clone M2).	anti-mCherry suggested: None anti-alpha Tubulin, suggested: None anti-FLAG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Affinity Purification of GFP-tagged constructs: The day prior to transient transfection, 10-cm plates were seeded with 4×106 293T cells and grown overnight to approximately 50% confluency.	293T suggested: None
   Software and Algorithms
   Sentences	Resources
   Multiplex amplicon sequencing libraries were constructed using Swift Biosciences’ SARS-CoV-2 Multiplex Primer Pool and Normalase Amplicon kit and sequenced on a 2 × 300 bp run on an Illumina MiSeq.	Swift Biosciences’ suggested: None
   Sequencing reads were quality- and adapter-trimmed using Trimmomatic v0.38 (ILLUMINACLIP:TruSeq3-PE-SNAP.fa:2:30:10:1:true LEADING:3 TRAILING:3 SLIDINGWINDOW:4:30 MINLEN:75) (39) and aligned to the SARS-CoV-2 reference genome (NC_045512.2) using BBMap version 38.70 (sourceforge.net/projects/bbmap/)	Trimmomatic suggested: (Trimmomatic, RRID:SCR_011848) BBMap suggested: (BBmap, RRID:SCR_016965)
   Sequence reads were then clipped of synthetic PCR primers using Primerclip (https://github.com/swiftbiosciences/primerclip) and the final sequence alignment was visualized in Geneious version 11.1.4 (40).	Geneious suggested: (Geneious, RRID:SCR_010519)
   The genetic relatedness of these strains was assessed by aligning the genomes of these strains as well as 110 other global clinical SAR-CoV-2 strains using MAFFT v7.453 (41).	MAFFT suggested: (MAFFT, RRID:SCR_011811)
   A phylogenetic tree was generated using RAxML version 8.2.11 (42) and visualized with R (version 3.6.1) using the ggtree package (43).	RAxML suggested: (RAxML, RRID:SCR_006086)
   mCherry fluorescence intensities were measured with ImageJ v1.53a by an individual blinded to experimental design.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Data Availability: Sequencing reads and genome assemblies are available under NCBI BioProject PRJNA610428.	BioProject suggested: (NCBI BioProject, RRID:SCR_004801)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Our study has a number of limitations. We relied on cellular overexpression systems of ORF6 which rely on nuclear export, making study of a likely nuclear export inhibition factor difficult. As such, our results may not perfectly reflect the degree to which host mRNA nuclear export and protein expression is downregulated during SARS-CoV-2 infection, which does not rely on nuclear export for ORF6 expression. More comparative work between SARS-CoV-2 and SARS-CoV ORF6 is needed in the context of viral replication. It would be intriguing to swap ORF6 between SARS-CoV and SARS-CoV-2 isolates to test the hypothesis that ORF6 is the major determinant of interferon antagonization and delayed symptom onset in animal models of SARS-CoV-2. Furthermore, additional work is required to understand the degree of mRNA export inhibition in both viruses. In summary, our results demonstrate the accessory protein ORF6 of SARS-CoV-2 strongly inhibits reporter protein expression and imprisons mRNA in the nucleus via its interactions with the mRNA nuclear export factor Rae1 and the nuclear pore complex component Nup98. As ORF6 is a major interferon antagonist in Sarbecoviruses, differences in ORF6 sequence content may be major determinants of differences in clinical presentation among these viruses that so clearly have the world’s attention.

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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.
   • No funding statement was detected.
   • No protocol registration statement was detected.

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3. 

   Version published to 10.1101/2020.08.03.234559 on bioRxiv

   Aug 3, 2020