Identification and differential usage of a host metalloproteinase entry pathway by SARS-CoV-2 Delta and Omicron

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1. Version published to 10.1016/j.isci.2022.105316

   Nov 1, 2022
2. 

   ScreenIT

   Feb 25, 2022

   SciScore for 10.1101/2022.02.19.481107: (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
   The monoclonal antibodies SARS-CoV-1/SARS-CoV-2 Spike Protein S2 (1A9) and SARS-CoV-1/SARS-CoV-2 Nucleocapsid (6H3) were purchased from ThermoFisher Scientific.	SARS-CoV-1/SARS-CoV-2 Nucleocapsid ( 6H3 ) suggested: None
   The rabbit polyclonal Anti-GAPDH antibody were purchased from Abcam.	Anti-GAPDH suggested: None
   The rabbit polyclonal Anti-HIV-1 p24 antibody was purchased from MilliporeSigma.	Anti-HIV-1 suggested: None
   The mouse anti-N protein antibody (clone 1C7) was purchased from Bioss …

   SciScore for 10.1101/2022.02.19.481107: (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
   The monoclonal antibodies SARS-CoV-1/SARS-CoV-2 Spike Protein S2 (1A9) and SARS-CoV-1/SARS-CoV-2 Nucleocapsid (6H3) were purchased from ThermoFisher Scientific.	SARS-CoV-1/SARS-CoV-2 Nucleocapsid ( 6H3 ) suggested: None
   The rabbit polyclonal Anti-GAPDH antibody were purchased from Abcam.	Anti-GAPDH suggested: None
   The rabbit polyclonal Anti-HIV-1 p24 antibody was purchased from MilliporeSigma.	Anti-HIV-1 suggested: None
   The mouse anti-N protein antibody (clone 1C7) was purchased from Bioss Antibodies, and the rabbit anti-SARS-CoV-2 spike protein (clone 007) antibody, was purchased from Sino Biological.	anti-N protein suggested: None
   protein primary antibody and an anti-mouse IgG HRP secondary antibody in conjunction with SIGMAFAST™ OPD developing solution	anti-mouse IgG suggested: None
   In parallel and after blocking, the second plate was incubated for one hour with a primary antibody solution formulated in PBS + 1% non-fat milk containing both mouse anti-N protein (1 μg/mL, clone 1C7) and rabbit anti-SARS-CoV-2 spike protein (1:500 dilution, clone 007) antibodies.	anti-N protein ( 1 suggested: None anti-SARS-CoV-2 spike protein suggested: None
   donkey anti-rabbit IgG Alexa Fluor Plus 594 (2 μg/mL, Invitrogen) antibodies and DAPI (1:1000, Millipore Sigma) in PBS + 0.5% BSA consisting of.	anti-rabbit IgG suggested: (Thermo Fisher Scientific Cat# A48284, RRID:AB_2896348)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines, inhibitors, and antibodies: HEK293T (ATCC), HEK293T-ACE2 (kind gift of Hyeryun Choe, Scripps Research), HT1080 cells (ATCC) and Calu3 (ATCC) were cultured in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Sigma), 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.3 mg/mL L-glutamine.	Calu3 suggested: None
   Fusion assays: For the syncytium formation assay HEK293T and HEK293T-Ace2 cells were seeded in 24-well plates and grown to approximately 80% confluency.	HEK293T-Ace2 suggested: None
   Cell-cell fusion assay with soluble ACE2, effector HEK293T cells were transiently transfected with plasmid DNA encoding mCherry, and SARS-CoV2 spike and target HEK293T cells were transiently transfected with plasmid DNA encoding LTR-GFP, TMPRSS2 or pCAGGS.	HEK293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
   Gelatin zymography: HEK293T, HEK293T-ACE2, Calu3 and HT1080 cells were analyzed for MMP2 and MMP9 activity through zymographic analysis.	HT1080 suggested: CLS Cat# 300216/p517_HT-1080, RRID:CVCL_0317)
   HT1080-ACE2 cells were cultured in DMEM supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), HEPES, L-Glutamine (0.3 mg/mL), 10% FBS (all from Thermo Fisher Scientific) and puromycin (1 μg/mL, InvivoGen).	HT1080-ACE2 suggested: None
   Twenty-four hours before infection, 2.5×104 HT1080 ACE2 cells were seeded per well of duplicate 96 well plates in puromycin-deficient DMEM and cultured overnight (37°C/5% CO2) for cell monolayer to adhere.	HT1080 ACE2 suggested: None
   Recombinant DNA
   Sentences	Resources
   HT1080 cells stably expressing ACE2 were generated by infection with lentiviral particles generated with psPAX2, pMDG and pLENTI_hACE2_PURO (gift from Raffaele De Francesco	psPAX2 suggested: RRID:Addgene_12260) pMDG suggested: None pLENTI_hACE2_PURO suggested: RRID:Addgene_155295)
   The full gene, untagged or with a N-terminal FLAG tag, was reconstituted by Gibson assembly, amplified by PCR, and cloned in pCAGGS.	pCAGGS suggested: RRID:Addgene_127347)
   Software and Algorithms
   Sentences	Resources
   Time-course imaging of the syncytia formation was performed using an Incucyte-Zoom (EssenBioscience), and images were analyzed in imageJ to measure the percentage of green surface area over background.	imageJ suggested: (ImageJ, RRID:SCR_003070)
   Statistical analyses were performed with GraphPad Prism 9.	GraphPad Prism 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: 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 10.1101/2022.02.19.481107v1 on bioRxiv

   Feb 22, 2022