SARS-CoV-2 Spike evolution influences GBP and IFITM sensitivity

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   Version published to 10.1101/2022.03.07.481785v2 on bioRxiv

   May 17, 2022
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   ScreenIT

   Mar 10, 2022

   SciScore for 10.1101/2022.03.07.481785: (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
   Twenty micrograms of cell lysate and an equal volume of purified virus were separated by SDS-PAGE, transferred to nitrocellulose membrane and membranes probed using the following primary antibodies: anti-SARS-CoV Spike (100% overlap with the SARS-CoV-2 epitope) (Invitrogen, PA1-41165, 1:1000)	anti-SARS-CoV suggested: None SARS-CoV-2 epitope suggested: None PA1-41165 suggested: (Thermo Fisher Scientific Cat# PA1-41165, RRID:AB_1087210)
   , anti SARS-CoV nucleoprotein (N) monoclonal antibody …

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   SciScore for 10.1101/2022.03.07.481785: (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
   Twenty micrograms of cell lysate and an equal volume of purified virus were separated by SDS-PAGE, transferred to nitrocellulose membrane and membranes probed using the following primary antibodies: anti-SARS-CoV Spike (100% overlap with the SARS-CoV-2 epitope) (Invitrogen, PA1-41165, 1:1000)	anti-SARS-CoV suggested: None SARS-CoV-2 epitope suggested: None PA1-41165 suggested: (Thermo Fisher Scientific Cat# PA1-41165, RRID:AB_1087210)
   , anti SARS-CoV nucleoprotein (N) monoclonal antibody (clone CR3009, gift from Laura McCoy, 0.5 μg/ml), anti-HA-tag (Biolegend, 16B12, 1:2000)	anti SARS-CoV nucleoprotein ( N suggested: None anti-HA-tag suggested: None
   Primary antibodies were incubated overnight at 4°C with agitation and detected with fluorescent secondary antibodies: anti-Rabbit IgG (IRDye 800CW, Abcam, ab216773, 1:10,000), anti-Mouse IgG (IRDye 680RD, Abcam, ab216776, 1:10,000) and anti-Human IgG (IRDye 800CW, Licor, 925-68078, 1:10,000), and imaged with an Odyssey Cxl Infrared Imager (Licor).	anti-Rabbit IgG suggested: None anti-Mouse IgG suggested: None ab216776 suggested: None anti-Human IgG suggested: (LI-COR Biosciences Cat# 925-68078, RRID:AB_2814915)
   Cells were then washed and permeabilised with Perm buffer (Biolegend) for 5 min at RT and stained with primary antibodies (20 min at RT): anti-HA-tag-APC (Biolegend, 16B12, 1:150), anti-Gag-PE (Beckam Coulter, KC57, 1:250).	anti-HA-tag-APC suggested: None anti-Gag-PE suggested: None KC57 suggested: (Beckman Coulter Cat# 6604667, RRID:AB_1575989)
   SARS-CoV-2 infection was detected by intracellular staining for nucleoprotein (N) using anti-SARS-CoV N monoclonal antibody (human, clone CR3009, gift from Laura McCoy, 1 μg/ml) and secondary anti-Human IgG-AF488 (Jackson labs, 1:400).	anti-SARS-CoV N suggested: None anti-Human IgG-AF488 suggested: (SouthernBiotech Cat# 2040-30, RRID:AB_2795650)
   For analysis of cell surface spike infection, 293T cells were harvested with 5 mM EDTA (Sigma), washed, stained with Zombie NIR and anti-SARS-CoV-2 Spike antibody from human convalescent sera (NIBSC, 20/130 1:200) on ice for 30 min.	anti-SARS-CoV-2 suggested: None
   Primary antibody incubation was carried out for 1h at RT with rabbit anti-HA antibody (H6908, Sigma-Aldrich) to label IFITM1, IFITM2 and IFITM3 and mouse anti-CD63 (IB5, a gift from M. Marsh, UCL) to label endogenous CD63.	anti-HA suggested: (Sigma-Aldrich Cat# H6908, RRID:AB_260070) IFITM1 suggested: None IFITM2 suggested: None IFITM3 suggested: None IB5 suggested: None CD63 suggested: None
   Primary antibodies were detected with secondary anti-rabbit AlexaFluor-488 and anti-mouse AlexaFluor-568 conjugates (Jackson Immuno Research) for 1h.	anti-rabbit suggested: None anti-mouse suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cells: HEK293T/17 cells (abbreviated herein as 293T cells) were obtained from American Type Culture Collection (ATCC,	293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
   Vero.E6 cells were obtained from the National Institute for Biological Standards and Control (NIBSC).	Vero.E6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Vero.E6-TMPRSS2 cells (stable cell line expressing TMPRSS2 under G418 selection) were a gift from Mala Maini (UCL).	Vero.E6-TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   HeLa-TZM-bl cells (expressing luciferase and beta-galactosidase under the control of HIV-1 LTR) were obtained from the Centre for AIDS Reagents (CFAR).	HeLa-TZM-bl suggested: None
   Pseudovirus infection: Target cells were seeded into white 96-well plates 24h before infection (Caco2, Vero.E6 and Vero.E6-TMPRSS2 cells seeded at 1.5×104 cells/well, 293T-ACE2, HeLa-ACE2 and HeLa-TZMbl cells seeded at 1×104 cells/well).	HeLa-ACE2 suggested: JCRB Cat# JCRB1845, RRID:CVCL_B3LW) HeLa-TZMbl suggested: None
   IFITM inhibition assay: Caco2 cells, WT or stably expressing IFITM1/2/3 (described above), were infected with indicated spike PVs and luciferase expression was measured 48 h post-infection as described above.	Caco2 suggested: None
   Infection of Caco2-IFITM1/2/3 cells with live SARS-CoV-2 virus was done as described above.	Caco2-IFITM1/2/3 suggested: None
   Recombinant DNA
   Sentences	Resources
   Plasmids: SARS-CoV-2 Spike expression vectors were originally synthesised by Genewiz and subcloned into pCDNA3.1+ vector.	pCDNA3.1+ suggested: RRID:Addgene_117272)
   Lentiviral backbone packaging plasmid (expressing HIV Gag, Pol, Tat and Rev) p8.91 and the reporter plasmid encoding luciferase gene pCSLW were a gift from Greg Towers (UCL).	p8.91 suggested: None
   Pseudovirus production: Spike pseudoviruses (PVs) were made by co-transfection of spike, p8.91 and pCSLW plasmids as described previously (Dicken et al., 2021).	pCSLW suggested: None
   For HIV-1 Env PV, 293T cells were seeded as described above and transfected with 240 ng p8.91, 240 ng pCSLW,120 ng pSVIII-JRFL Env and indicated doses of GBP or empty vector control.	pCSLW,120 suggested: None pSVIII-JRFL suggested: None
   Software and Algorithms
   Sentences	Resources
   Plasmids: SARS-CoV-2 Spike expression vectors were originally synthesised by Genewiz and subcloned into pCDNA3.1+ vector.	Genewiz suggested: (GENEWIZ, RRID:SCR_003177)
   Scale bars and RGB composite images were created in in FIJI ImageJ software package (Schindelin et al., 2012).	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Statistical Analysis: Statistical significance was calculated using Prism 9 (GraphPad Prism) using indicated statistical tests and significance was assumed when P < 0.05.	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: 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 10.1101/2022.03.07.481785v1 on bioRxiv

   Mar 7, 2022