Highly Efficient SARS-CoV-2 Infection of Human Cardiomyocytes: Spike Protein-Mediated Cell Fusion and Its Inhibition

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1. Version published to 10.1128/jvi.01368-21

   Nov 23, 2021
2. 

   ScreenIT

   Aug 4, 2021

   SciScore for 10.1101/2021.07.30.454437: (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	Bright-field microscopy images were taken at 10x magnification from randomly chosen areas of each culture dish.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antibodies and reagents for immunocytochemistry included: ACTC1 (Actin α-sarcomeric mouse mAb clone 5C5 (Sigma)	ACTC1 suggested: None Actin suggested: None
   Nucleocapsid clone 1C7 (Bioss Antibodies), ACE2 goat polyclonal Ab (R&D Systems) and ATP2A2/SERCA2 rabbit polyclonal Ab (Cell Signaling).	ACE2 suggested: None ATP2A2/SERCA2 suggested: None
   The S-proteins were visualized on an immunoblot using …

   SciScore for 10.1101/2021.07.30.454437: (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	Bright-field microscopy images were taken at 10x magnification from randomly chosen areas of each culture dish.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antibodies and reagents for immunocytochemistry included: ACTC1 (Actin α-sarcomeric mouse mAb clone 5C5 (Sigma)	ACTC1 suggested: None Actin suggested: None
   Nucleocapsid clone 1C7 (Bioss Antibodies), ACE2 goat polyclonal Ab (R&D Systems) and ATP2A2/SERCA2 rabbit polyclonal Ab (Cell Signaling).	ACE2 suggested: None ATP2A2/SERCA2 suggested: None
   The S-proteins were visualized on an immunoblot using the anti-S specific monoclonal antibody 1A9 (GeneTex, GTX632604; 1:2000 dilution) which binds the S2 subunit of SARS CoV and SARS-CoV-2 S-proteins.	anti-S suggested: None GTX632604 suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
   An anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody was used to reveal the bands.	anti-mouse suggested: None
   MERS S-protein was detected using a monoclonal anti-FLAG M2-HRP conjugated antibody (SIGMA, A8592 @ 1:2000) which bound to a C-terminal FLAG-tag.	anti-FLAG M2-HRP suggested: None FLAG-tag suggested: None
   The expression of the mEmerald tag was verified using a polyclonal anti-GFP antibody (Abcam, ab290 @ 1:5000).	anti-GFP suggested: (Abcam Cat# ab290, RRID:AB_303395)
   Experimental Models: Cell Lines
   Sentences	Resources
   SARS-CoV-2 infection of hiPSC-CM: SARS-CoV-2/UW-001/Human/2020/Wisconsin (UW-001) was isolated from a mild case in February 2020 and passaged in VeroE6 cells expressing TMPRSS2.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Virus titration: SARS-CoV-2 infectious virus produced by hiPSC-CM was titered by plaque-forming assay done in confluent Vero E6/TMPRSS2 cells.	Vero E6/TMPRSS2 suggested: None
   After initial exposure, the Vero/TMPRSS2 cells were washed three times to remove unbound virus and the media was replaced with 1.0% methylcellulose-media.	Vero/TMPRSS2 suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
   Cell-cell fusion was followed for 72-hours (for Vero cells) and 5 days for hiPSC-CMS with media and inhibitor refreshed on day-3.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   Fluorescence-activated cell sorting: To determine S-protein cell surface expression levels, HeLa cells (8 × 105 in a 6-well plate) were transfected with the indicated S-protein expression plasmids (2 µg using GeneJuice transfection reagent).	HeLa suggested: None
   Recombinant DNA
   Sentences	Resources
   Plasmids and mutagenesis: The codon-optimized SARS-CoV2 S-protein gene (YP_009724390) was synthesized by Genewiz in a pUC57-Amp plasmid (kindly provided by M. Barry).	pUC57-Amp suggested: None
   The S-protein coding sequence was cloned into a pCG mammalian expression plasmid [52] using unique restriction sites BamHI and SpeI.	pCG suggested: RRID:Addgene_51476)
   Software and Algorithms
   Sentences	Resources
   Bioinformatics and data analysis: The quality of the raw RNA-seq data was assessed by fastqp v0.20.1 [44], and quality reads were filtered and aligned against human genome (hg19) using STAR alignment (v2.7.8a) [45] in galaxy platform (https://usegalaxy.org).	STAR suggested: (STAR, RRID:SCR_004463)
   The aligned reads were counted using htseq-count v0.9.1 [46] and 0.5 read counts per million (CPM) in at least two samples was used as an expression threshold.	htseq-count suggested: (htseq-count, RRID:SCR_011867)
   Trimmed mean of M values normalized (TMM) [47] and log2 transformed data was used for plotting heatmaps and differential analysis in limma [48].	limma suggested: (LIMMA, RRID:SCR_010943)
   For the detection of viral transcripts, quality filtered reads were aligned against SARS-CoV-2 genome (MT039887.1) using BWA-MEM v0.7.17.1 (	BWA-MEM suggested: (Sniffles, RRID:SCR_017619)
   Alignment summary statistics was computed using samtools idxstats v2.0.3 [49].	samtools suggested: (SAMTOOLS, RRID:SCR_002105)
   The raw RNA-seq data from this study are available at Gene Expression Omnibus with accession number xxxx [to be added].	Gene Expression Omnibus suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
   Plasmids and mutagenesis: The codon-optimized SARS-CoV2 S-protein gene (YP_009724390) was synthesized by Genewiz in a pUC57-Amp plasmid (kindly provided by M. Barry).	Genewiz suggested: (GENEWIZ, RRID:SCR_003177)
   ANOVA statistical analysis was carried out using GraphPad Prism software.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   After three washes with FACS wash buffer, cells were fixed in 4% paraformaldehyde and analyzed with a FACSCalibur (BD Biosciences, San Jose, CA) cytometer and FlowJo software (Tree Star Inc.	FACSCalibur suggested: None FlowJo suggested: (FlowJo, RRID:SCR_008520)

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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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 34 and 37. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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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.
   • No funding statement was detected.
   • 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 10.1101/2021.07.30.454437 on bioRxiv

   Jul 30, 2021