LY6E impairs coronavirus fusion and confers immune control of viral disease
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SciScore for 10.1101/2020.03.05.979260: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All procedures used in this study complied with federal and institutional guidelines enforced by the UTSW Institutional Animal Care and Use Committee (IACUC). Randomization not detected. Blinding Slides were analyzed by an independent pathologist (UTSW Animal Resource Center) who was blinded to experimental conditions. Power Analysis not detected. Sex as a biological variable In vivo infection, viral titers, liver ALT, and liver histology: Six to twelve-week-old female and male mice were injected intraperitoneally with MHV-A59 diluted in PBS to the indicated titers or PBS for a mock infection control. Cell Line Authentication not detected. Table 2: Resources
… SciScore for 10.1101/2020.03.05.979260: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All procedures used in this study complied with federal and institutional guidelines enforced by the UTSW Institutional Animal Care and Use Committee (IACUC). Randomization not detected. Blinding Slides were analyzed by an independent pathologist (UTSW Animal Resource Center) who was blinded to experimental conditions. Power Analysis not detected. Sex as a biological variable In vivo infection, viral titers, liver ALT, and liver histology: Six to twelve-week-old female and male mice were injected intraperitoneally with MHV-A59 diluted in PBS to the indicated titers or PBS for a mock infection control. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 24 hours post-infection, cells were dissociated using Accumax (Sigma-Aldrich), fixed in 1% PFA, permeabilized per manufacturer’s protocol (BD Cytofix/Cytoperm), and stained for nucleoprotein (1:500) and a goat anti-mouse AlexaFluor488-conjugated secondary antibody (1:2000). anti-mousesuggested: NoneIHIC and splenocytes (4 × 105) from infected or mock-infected mice were stained with a fluorescent viability dye (Ghostdye Violet 450, Tonbo Biosciences), treated with anti-CD16/CD32 (Tonbo Biosciences), stained with antibodies that recognize surface lineage markers, and then fixed in 1% PFA/PBS. anti-CD16/CD32suggested: NoneExperimental Models: Cell Lines Sentences Resources HCoV-229E viruses were propagated on Huh-7 cells, MERS-CoV and SARS-CoV were propagated on VeroB4 cells, HCoV-OC43 was propagated on HCT-8 cells38, and MHV stocks were propagated on 17Cl1 cells. Huh-7suggested: NoneVeroB4suggested: CCLV Cat# CCLV-RIE 1146, RRID:CVCL_1912)For testing the virus panel against LY6E, 1 × 104 stable LY6E expressing or control Huh7.5 cells were seeded in a 96-well plate. Huh7.5suggested: RRID:CVCL_7927)Briefly, 6 × 105 293LTV cells were seeded in a 6-well plate and transfected using Lipofectamine 2000 (Invitrogen) which was complexed with DNA plasmids driving the expression of either VSV G protein (positive control), the respective CoV S proteins, or the fluorophore mCherry (negative control). 293LTVsuggested: RRID:CVCL_JZ09)The empty expression plasmid (pCAGGS-mCherry) as well as a construct encoding for VSV G served as negative and positive control, respectively. 2 × 105 stable LY6E expressing or empty control Huh7 cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) with a plasmid encoding the second half of the split-luciferase protein (Rluc8155-156 DSP8-11). Huh7suggested: NoneGeneration of recombinant VSV vector driving CoV S protein expression (VSV*ΔG(CoV)): Following extraction of total RNA from MERS-CoV (strain EMC) infected VeroE6 cells, the cDNA encoding the MERS-CoV S protein was generated by reverse transcription (RevertAid Premium Reverse Transkriptase, ThermoScientific). VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)For heterologous cell-cell fusion assay, BHK-21 cells were infected with VSV*ΔG(CoV S) at a MOI of 1. BHK-21suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)Viral titers in liver and spleen were determined from frozen organs after weighing, homogenization, and plaque assay on L929 cells. L929suggested: ECACC Cat# 86032004, RRID:CVCL_4238)Experimental Models: Organisms/Strains Sentences Resources Mice: Ly6etm1a ES cells were obtained from the EUCOMM consortium58 and microinjected into C57BL/6J blastocysts by the UTSW Transgenic Technology Center. C57BL/6Jsuggested: RRID:IMSR_JAX:000664)Ly6etm1a/+ mice were crossed with FLPe-expressing mice (B6N.129S4-Gt(ROSA)26Sortm1(FLP1)Dyn/J, #16226, Jackson Laboratories) to obtain Ly6efl/+ offspring, which were bred to homozygosity. Ly6etm1a/+suggested: NoneB6N.129S4-Gt(ROSA)26Sortm1 ( FLP1)Dyn/Jsuggested: NoneLy6efl/+suggested: NoneConditional Ly6efl/fl mice were bred to Vav1-iCre transgenic mice (B6.Cg-Commd10Tg(Vav1-icre)A2Kio/J, #008610, Jackson Laboratories) to obtain Ly6eΔHSC/+ (Ly6efl/+; Vav1-iCre) offspring. Ly6efl/flsuggested: NoneVav1-iCresuggested: NoneB6.Cg-Commd10Tg(Vav1-icre)A2Kio/Jsuggested: RRID:IMSR_JAX:008610)Ly6eΔHSC/+ mice were bred to obtain Ly6eΔHSC (Ly6efl/fl; Vav1-iCre) offspring, which harbor a deletion of exon 3 and 4 in hematopoietic stem cells (HSC). Ly6eΔHSC/+suggested: NoneLy6efl/fl; Vav1-iCresuggested: NoneSoftware and Algorithms Sentences Resources Data was analyzed with FlowJo Software (Treestar). FlowJosuggested: (FlowJo, RRID:SCR_008520)Statistical analysis: Differences in data were tested for significance using GraphPad Prism v8.3.1 for Windows (GraphPad). GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)The RNAseq data discussed in this publication have been deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (GSE146074). Gene Expression Omnibussuggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)Results from OddPub: Thank you for sharing your data.
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: Please consider improving the rainbow (“jet”) colormap(s) used on page 33. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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- No protocol registration statement was detected.
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