VPS29 Exerts Opposing Effects on Endocytic Viral Entry
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Abstract
These data identify a host pathway by which VPS29 and associated factors control the endosomal environment in a manner that influences susceptibility to viral infection. This pathway could serve as a pharmaceutical target for intervention in zoonotic viral diseases, including those caused by coronaviruses, influenza viruses, and filoviruses, all of which are pandemic threats.
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SciScore for 10.1101/2021.08.06.455441: (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 Briefly, cells were blocked with 5.0% fetal bovine serum in PBS and permeabilized with 0.5% Saponin before a 30 minutes incubation with: HCoV-OC43: Anti-Coronavirus Group Antigen Antibody, nucleoprotein of OC-43 (1:1000, Sigma MAB9013); Anti-Coronavirus Group Antigen Antibody , nucleoprotein of OC-43suggested: None48 hours post transduction, cells were fixed in 4% PFA, permeabilized with 0.1% triton, blocked with FBS and stained for V5 (invitrogen cat# 46-0705, 1:1000) and antibody … SciScore for 10.1101/2021.08.06.455441: (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 Briefly, cells were blocked with 5.0% fetal bovine serum in PBS and permeabilized with 0.5% Saponin before a 30 minutes incubation with: HCoV-OC43: Anti-Coronavirus Group Antigen Antibody, nucleoprotein of OC-43 (1:1000, Sigma MAB9013); Anti-Coronavirus Group Antigen Antibody , nucleoprotein of OC-43suggested: None48 hours post transduction, cells were fixed in 4% PFA, permeabilized with 0.1% triton, blocked with FBS and stained for V5 (invitrogen cat# 46-0705, 1:1000) and antibody conjugate AF-488 Goat anti-Mouse IgG (H+L) (Thermo, 1:1000). V5suggested: Noneanti-Mouse IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources To generate lentiviral preparations of the Brunello library, 293T cells (6 × 106 cells per 10 cm dish) were transfected with 6µg lentiCRISPRv2-Brunello, 6µg NL-gagpol, and 1.2 µg VSV-G using PEI. 48 hours post transfection, supernatants were pooled and concentrated using Amicon Ultra Centrifugal Filters 293Tsuggested: NoneBriefly, 10-fold serial dilutions (from 10−1 to 10−6) were used to transduce 40,000 A549 cells in a 24 well plate format. A549suggested: NoneThereafter, in triplicates with 8×106 cells per flask, A549-Brunello cells were infected or not with HCoV-OC43 at an MOI of 0.1, and passaged for 7 days until >95% infection-induced cell death occurred. A549-Brunellosuggested: NoneRecombinant DNA Sentences Resources The next day, 7.5 µg pHIV-1NL4-3 ΔEnv-NanoLuc and 2.5 µg indicated CoV spike plasmid were transfected using PEI. pHIV-1NL4-3suggested: NoneSoftware and Algorithms Sentences Resources Pathway Analysis of screen hits: All 34 candidate genes were searched using the STRING database (https://string-db.org) for functional enrichment of protein-protein interactions using default settings, except the minimum required interaction score was changed from medium confidence (0.400) to high confidence (0.700). STRINGsuggested: (STRING, RRID:SCR_005223)Quantification of fluorescence microscopy: For each cell, Regions Of Interest (ROIs) corresponding to labeled endosomes were defined using the freehand selection tool in Fiji. Fijisuggested: (Fiji, RRID:SCR_002285)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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 17 and 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.
- 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.
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