The P681H mutation in the Spike glycoprotein confers Type I interferon resistance in the SARS-CoV-2 alpha (B.1.1.7) variant

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1. 

   ScreenIT

   Nov 19, 2021

   SciScore for 10.1101/2021.11.09.467693: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Sample collection and studies were performed in accordance with the UK Policy Framework for Health and Social Care Research and with specific Research Ethics Committee approval (REC 20/SC/0310). IRB: Sample collection and studies were performed in accordance with the UK Policy Framework for Health and Social Care Research and with specific Research Ethics Committee approval (REC 20/SC/0310).
   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
   Membranes were blocked in milk prior to detection with specific antibodies: 1:1000 ACE2 …

   SciScore for 10.1101/2021.11.09.467693: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Sample collection and studies were performed in accordance with the UK Policy Framework for Health and Social Care Research and with specific Research Ethics Committee approval (REC 20/SC/0310). IRB: Sample collection and studies were performed in accordance with the UK Policy Framework for Health and Social Care Research and with specific Research Ethics Committee approval (REC 20/SC/0310).
   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
   Membranes were blocked in milk prior to detection with specific antibodies: 1:1000 ACE2 rabbit (Abcam, Ab108209),1:5000	ACE2 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cells and plasmids: HEK293T-17 (ATCC, CRL-11268™), Calu-3 (ATCC, HTB-55™), A549-ACE2, Vero-E6, Vero-E6-TMPRSS2 and A549-ACE2 expressing the individual IFITM proteins were cultured in DMEM (Gibco) with 10% FBS (Invitrogen) and 200μg/ml Gentamicin (Sigma), and incubated at 37°C, 5% CO2.	Calu-3 suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
   A549 stable cell lines expressing ACE2 (pMIGR1-puro), and IFITMs (pLHCX) were generated and selected as described previously (Winstone et al., 2021)	A549 suggested: None
   Passage and titration of SARS-CoV-2: PHE England strain 02/2020 was propagated in Vero-E6-TMPRSS2 cells and titre was determined by plaque assay (Winstone et al., 2021).	Vero-E6-TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Approximately 2.5 μg of the in vitro synthesized RNA was used to transfect ∼6 ×105 BHK-hACE2-N cells stably expressing the SARS-CoV-2 N and the human ACE2 genes(Rihn et al., 2021) using the MessengerMax lipofection kit (Thermo Scientific) as per the manufacturer’s instructions.	BHK-hACE2-N suggested: None
   Cells were then incubated until signs of viral replication (syncytia formation) became visible (usually after 2-3 days), at which time the medium was collected (P0 stock) and used further as a source of rescued virus to infect VERO E6 cells to generate P1 and P2 stocks.	VERO E6 suggested: RRID:CVCL_XD71)
   Generation of Clinical Viral Isolates: Viruses were isolated on Vero.E6 cells (ATCC CRL 1586™) from combined naso-oropharyngeal swabs submitted for routine diagnostic testing by real-time RT-PCR and shown to be from the B.1.1.7 (alpha) variant by on-site whole-genome sequencing (Oxford Nanopore Technologies, Oxford, UK) (Pickering et al., 2021)	Vero.E6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Viral RNA levels in cells or supernatants were measured 48 hours after infection by RT-qPCR. siRNA knockdown of IFITM2: A549-ACE2 cells were reverse transfected using 20pmol of Non-targeting siRNA (D-001206-13-20) or IFITM2 siRNA (M-020103-02-0010) and 1μL of RNAi max (Invitrogen).	A549-ACE2 suggested: None
   Recombinant DNA
   Sentences	Resources
   A549 stable cell lines expressing ACE2 (pMIGR1-puro), and IFITMs (pLHCX) were generated and selected as described previously (Winstone et al., 2021)	pMIGR1-puro suggested: None
   Briefly, a set of overlapping cDNA fragments representing the entire genomes of SARS-CoV-2 Wuhan isolate (GenBank: MN908947.3) and the B.1.1.7 alpha variant were chemically synthesized and cloned into pUC57-Kan (Bio Basic Canada Inc and Genewiz, respectively).	pUC57-Kan suggested: RRID:Addgene_123653)
   To generate Wuhan virus carrying the alpha variant spike, a mixture of the relevant synthetic cDNA fragments of the Wuhan and alpha variants was co-transformed with XhoI-BamHI-cut pEB2 into the Saccharomyces cerevisiae strain TYC1 (MATa, ura3-52, leu2Δ1, cyh2r, containing a knockout of DNA Ligase 4) (Gaida et al., 2011) that had been made competent for DNA uptake using the LiCl2-based Yeast transformation kit (YEAST1-1KT, Merck).	pEB2 suggested: RRID:Addgene_104001)
   Software and Algorithms
   Sentences	Resources
   Viruses were sequenced using Oxford Nanopore as previously described(da Silva Filipe et al., 2021).	Oxford Nanopore suggested: (Oxford Nanopore Technologies, RRID:SCR_003756)

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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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2. 

   Version 1 published on bioRxiv

   Nov 10, 2021