SARS-CoV-2 spike N-terminal domain modulates TMPRSS2-dependent viral entry and fusogenicity
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
- Evaluated articles (ScreenIT)
Abstract
No abstract available
Article activity feed
-
-
SciScore for 10.1101/2022.05.07.491004: (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: All cells were regularly tested and are mycoplasma free. Table 2: Resources
Antibodies Sentences Resources For protein detection, the following antibodies were used: rabbit anti-SARS-CoV-2 S monoclonal antibody (PA1-41165; Thermofisher), mouse anti-SARS-CoV-2 S1 (MAB105403, R&D systems), rabbit anti-GAPDH polyclonal antibody (10494-1-AP; Proteintech), horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse IgG polyclonal antibody (Cell Signalling). anti-SARS-CoV-2 Ssuggested: Noneanti-SARS-CoV-2suggested: Noneanti-GAPDHsugg…SciScore for 10.1101/2022.05.07.491004: (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: All cells were regularly tested and are mycoplasma free. Table 2: Resources
Antibodies Sentences Resources For protein detection, the following antibodies were used: rabbit anti-SARS-CoV-2 S monoclonal antibody (PA1-41165; Thermofisher), mouse anti-SARS-CoV-2 S1 (MAB105403, R&D systems), rabbit anti-GAPDH polyclonal antibody (10494-1-AP; Proteintech), horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse IgG polyclonal antibody (Cell Signalling). anti-SARS-CoV-2 Ssuggested: Noneanti-SARS-CoV-2suggested: Noneanti-GAPDHsuggested: (Proteintech Cat# 10494-1-AP, RRID:AB_2263076)anti-rabbitsuggested: Noneanti-mouse IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Vero-ACE2/TMPRSS2 cells (a gift from Emma Thomson), Hela-ACE2 (a gift from James Voss) and A549-ACE2/TMPRSS2 (a gift from Massimo Palmarini were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and 1% PS. 293T (CRL-3216) and its derivative cell lines including 293TACE2ΔTMPRSS2, 293T-TMPRSS2 and 293T-GFP11 have been described previously (Papa et al., 2021). Vero-ACE2/TMPRSS2suggested: NoneIn brief, 293T cells were transfected with a mixture of 11ul of Fugene HD, 1μg of pCDNAΔ19 spike, 1ug of p8.91 HIV-1 gag-pol expression vector and 1.5μg of pCSFLW (expressing the firefly luciferase reporter gene with the HIV-1 packaging signal). 293Tsuggested: NoneWestern blot: For cell lysates, 293 cells were washed and lysed in lysis buffer (Cell Signalling) and lysates were diluted with 4 × sample buffer (Biorad) and boiled for 10 m before subjected to western blotting. 293suggested: NoneDrug assay: A549-ACE2-TMPRSS2 (A549-A2T2) cells or human airway organoids were either E64D (Tocris) or camostat (Sigma-Aldrich) treated for 2 hours at each drug concentration before the addition of a comparable amount of input viruses pseudotyped with Delta, Kappa or chimeras (approx. 100,000 RLU). A549-A2T2suggested: NoneBriefly, 293T GFP11 and Vero-GFP1-10 cells were seeded at 80% confluence in a 1:1 ratio in 48 multiwell plate the day before. Vero-GFP1-10suggested: NoneRecombinant DNA Sentences Resources Airway organoids were cultured in 48-well plate and were passaged every 2 weeks as previously reported (Meng et al., 2022). pCDNA_SARS_CoV2_D416G_S WT, Delta, Kappa, Omicron BA.1 and Omicron BA. pCDNA_SARS_CoV2_D416G_Ssuggested: NoneAmino acid substitutions in the Delta NTD (D142G, G156E and repair of 157F and 158R) were introduced into the pCDNA_SARS-CoV-2_ D614G_Delta_S plasmid using the QuikChange Lightning Site-Directed Mutagenesis kit, following the manufacturer’s instructions (Agilent Technologies). pCDNA_SARS-CoV-2_ D614G_Delta_Ssuggested: NoneIn brief, 293T cells were transfected with a mixture of 11ul of Fugene HD, 1μg of pCDNAΔ19 spike, 1ug of p8.91 HIV-1 gag-pol expression vector and 1.5μg of pCSFLW (expressing the firefly luciferase reporter gene with the HIV-1 packaging signal). pCDNAΔ19suggested: NonepCSFLWsuggested: NoneSoftware and Algorithms Sentences Resources The raw readings (in relative light unit (RLU)) were then normalised with the SG-PERT and plotted using GraphPad Prism. PV SG-PERT: The SARS-CoV2 spike-pseudotyped viruses containing supernatants were standardised using a SYBR Green-based product-enhanced PCR assay (SG-PERT) as described previously (Pizzato et al., 2009). GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)The cleavage ratio of S1 or S2 to FL in virions was determined by densitometry using ImageJ (NIH). ImageJsuggested: (ImageJ, RRID:SCR_003070)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).
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.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
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.
Results from scite Reference Check: We found no unreliable references.
-
