SARS-CoV-2 ORF6 disturbs nucleocytoplasmic trafficking to advance the viral replication

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   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2021.02.24.432656: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: The animal experiments, and the study protocol were approved by the Institutional Committee of Laboratory Animal Experimentation of the Research Institute for Microbial Diseases, Osaka University (R02-08-0).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Four weeks-old male Syrian hamsters were purchased from SLC (Shizuoka, Japan).
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Using a rat isotyping kit the MAb 8B10 was found to be an IgG 1 (k) antibody subtype.	antibody subtype. suggested: None
   The monoclonal antibody against SARS-CoV-2 NP (3A9 clone) was generated by …

   SciScore for 10.1101/2021.02.24.432656: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: The animal experiments, and the study protocol were approved by the Institutional Committee of Laboratory Animal Experimentation of the Research Institute for Microbial Diseases, Osaka University (R02-08-0).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Four weeks-old male Syrian hamsters were purchased from SLC (Shizuoka, Japan).
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Using a rat isotyping kit the MAb 8B10 was found to be an IgG 1 (k) antibody subtype.	antibody subtype. suggested: None
   The monoclonal antibody against SARS-CoV-2 NP (3A9 clone) was generated by Cell Engineering Corporation (Osaka, Japan).	SARS-CoV-2 NP suggested: None 3A9 suggested: None
   Importin β1, p10/NTF2, and GDP-bound Ran were purified as previously described 47, 49. Antibodies: The following primary antibodies were used in this studies: Phospho-STAT1 (Tyr701) (#9167 [58D6], Cell Signaling Technology (CST) Inc.	Phospho-STAT1 ( Tyr701 ) ( #9167 [ 58D6] , Cell Signaling Technology ( CST ) Inc . suggested: None
   Horseradish peroxidase (HRP)-conjugated anti-rabbit (#111-035-003), anti-mouse (#115-035-003), or anti-rat (#112-035-003) secondary antibodies (Jackson ImmunoResearch Inc. West Grove, PA, USA) were used for western blotting.	anti-rat suggested: (Jackson ImmunoResearch Labs Cat# 112-035-003, RRID:AB_2338128)
   The secondary antibodies used for indirect immunofluorescence were as follows: Alexa Fluor Plus 488 conjugated anti-rabbit (A32731) or anti-mouse (A32723), and Alexa Fluor 594 conjugated anti-rabbit (A21207) or anti-mouse (A21203) (Invitrogen).	anti-rabbit ( A32731 suggested: None anti-mouse ( A32723 suggested: None anti-rabbit suggested: (Molecular Probes Cat# A-21207, RRID:AB_141637) anti-mouse suggested: (Molecular Probes Cat# A-21203, RRID:AB_141633)
   For the anti-Phospho-STAT1 antibody (58D6, Rabbit mAb, #9167, CST), cells were permeabilized with 100% methanol at −20 °C for 20 min and then blocked in 3% skim milk in PBS.	anti-Phospho-STAT1 suggested: None
   After treatment with 5% skim milk in PBS for 30 min at 25 °C, the sections were incubated with mouse anti-NP antibody (1:500, clone 3A9).	anti-NP suggested: (Thermo Fisher Scientific Cat# MA1-82295, RRID:AB_931436)
   Experimental Models: Cell Lines
   Sentences	Resources
   The cDNA of ORF6 was obtained from Vero-TMPRSS2 cells infected with SARS-CoV-2.	Vero-TMPRSS2 suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
   Cell culture and transfection: HeLa cells or HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen), containing 10% FBS (#10270, Gibco) at 37 °C in 5% CO2.	HEK293 suggested: None
   Indirect immunofluorescence: HeLa cells were cultured on 18 × 18 mm coverslips (Matsunami, Osaka, Japan) in 35-mm dishes (IWAKI) and incubated for 48 h at 37 °C in 5% CO2.	HeLa suggested: RRID:CVCL_IY74)
   Lentiviruses expressing three types of target sequences per gene were mixed, introduced into the Huh7 cells expressing the ACE2 receptor (Huh7-ACE2), and maintained in a culture medium supplemented with 1 µg/mL puromycin for 3 weeks.	Huh7 suggested: None
   For viral infection, sgControl (sgCtl) or sgKPNA2 Huh7-ACE2 cells were seeded into 24-well plates and incubated at 37 °C for 24 h.	Huh7-ACE2 suggested: None
   ACE2 and TMPRSS2 receptors were induced in HEK293-3P6C33 cells using tetracycline.	HEK293-3P6C33 suggested: None
   At 7-10 days post transfection, the culture medium containing progeny viruses (P0 virus) were passaged and amplified in VeroE6/TMPRSS2 cells.	VeroE6/TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Software and Algorithms
   Sentences	Resources
   Statistical Analysis: Data were analyzed with Prism 7.0 software (GraphPad Software, La Jolla, CA), and expressed as the mean ± standard deviation (SD).	Prism suggested: (PRISM, RRID:SCR_005375) 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 did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 41, 42, 45 and 47. 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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   Version published to 10.1101/2021.02.24.432656v1 on bioRxiv

   Feb 24, 2021