Spike Glycoprotein and Host Cell Determinants of SARS-CoV-2 Entry and Cytopathic Effects

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1. Version published to 10.1128/jvi.02304-20

   Feb 10, 2021
2. 

   ScreenIT

   Oct 26, 2020

   SciScore for 10.1101/2020.10.22.351569: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Cell lines: Human female embryonic kidney (HEK) 293T cells (ATCC) and COS-1 African green monkey male kidney fibroblasts (ATCC) were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 μg/ml penicillin-streptomycin (Pen-Strep) (Life Technologies).
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Samples were Western blotted with 1:2,000 dilutions of rabbit anti-SARS-Spike S1, mouse anti-SARS-Spike S1, rabbit anti-SARS-Spike S2, rabbit anti-p55/p24/p17 or mouse …

   SciScore for 10.1101/2020.10.22.351569: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Cell lines: Human female embryonic kidney (HEK) 293T cells (ATCC) and COS-1 African green monkey male kidney fibroblasts (ATCC) were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 μg/ml penicillin-streptomycin (Pen-Strep) (Life Technologies).
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Samples were Western blotted with 1:2,000 dilutions of rabbit anti-SARS-Spike S1, mouse anti-SARS-Spike S1, rabbit anti-SARS-Spike S2, rabbit anti-p55/p24/p17 or mouse anti-VSV-NP or a 1:10,000 dilution of mouse anti-β-actin as the primary antibodies.	anti-SARS-Spike suggested: None anti-VSV-NP suggested: None anti-β-actin suggested: None
   HRP-conjugated anti-rabbit or anti-mouse antibodies at a slightly lower dilution were used as secondary antibodies in the Western blots.	HRP-conjugated suggested: None anti-rabbit suggested: None anti-mouse suggested: None
   Beads were washed three times and samples were Western blotted with a mouse anti-S1 antibody.	anti-S1 suggested: None
   The pellets were suspended in 50 μl of 1X LDS buffer containing 100 mM DTT, and Western blotted using rabbit anti-S1, rabbit anti-S2 and anti-p55/p24/p17 antibodies.	anti-S2 suggested: None anti-p55/p24/p17 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines: Human female embryonic kidney (HEK) 293T cells (ATCC) and COS-1 African green monkey male kidney fibroblasts (ATCC) were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 μg/ml penicillin-streptomycin (Pen-Strep) (Life Technologies).	HEK suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
   COS-1 cells transiently expressing alpha-gal and SARS-CoV-2 S gp variants or HIV-1 envelope glycoproteins (AD8 or JR-FL strain) were used as effector cells in the cell-cell fusion assay.	COS-1 suggested: BCRC Cat# 60002, RRID:CVCL_0223)
   Lenti-x-293T cells were grown in DMEM with 10% heat-inactivated FBS supplemented with L-glutamine and Pen-Strep.	Lenti-x-293T suggested: None
   Briefly, the 293T-S-ACE2 cells were produced by transduction of the 293T-S cells with the K5659 recombinant lentivirus vector, which expresses human ACE2 (see below).	293T-S-ACE2 suggested: None
   The packaged K5659 lentivirus vector (60 μl volume) was incubated with 2×105 293T-S cells in DMEM, tumbling at 37°C overnight.	293T-S suggested: RRID:CVCL_LC70)
   S expression, processing and VLP incorporation: 293T cells were transfected to produce lentivirus or VSV VLPs pseudotyped with S gp variants, as described above.	293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
   For the SARS-CoV-2 S gp-expressing effector cells, 293T-ACE2 cells were used as target cells; for effector cells expressing the HIV-1JR-FL envelope glycoprotein, Cf2Th-CD4/CCR5 cells were used as target cells.	293T-ACE2 suggested: RRID:CVCL_YZ65)
   Software and Algorithms
   Sentences	Resources
   The concentrations of sACE2 and dilutions of sera that inhibited 50% of infection (the IC50 and ID50 values, respectively) were determined by fitting the data in five-parameter dose-response curves using GraphPad Prism 8 (GraphPad Software Inc.).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) 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: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
   • 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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3. 

   Version published to 10.1101/2020.10.22.351569v1 on bioRxiv

   Oct 23, 2020