SARS-CoV-2 activates ER stress and Unfolded protein response
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Abstract
Coronavirus disease-2019 (COVID-19) pandemic caused by the SARS-CoV-2 coronavirus infection is a major global public health concern affecting millions of people worldwide. The scientific community has joint efforts to provide effective and rapid solutions to this disease. Knowing the molecular, transmission and clinical features of this disease is of paramount importance to develop effective therapeutic and diagnostic tools. Here, we provide evidence that SARS-CoV-2 hijacks the glycosylation biosynthetic, ER-stress and UPR machineries for viral replication using a time-resolved (0-48 hours post infection, hpi) total, membrane as well as glycoproteome mapping and orthogonal validation. We found that SARS-CoV-2 induces ER stress and UPR is observed in Vero and Calu-3 cell lines with activation of the PERK-eIF2α-ATF4-CHOP signaling pathway. ER-associated protein upregulation was detected in lung biopsies of COVID-19 patients and associated with survival. At later time points, cell death mechanisms are triggered. The data show that ER stress and UPR pathways are required for SARS-CoV-2 infection, therefore representing a potential target to develop/implement anti-CoVID-19 drugs.
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SciScore for 10.1101/2021.06.21.449284: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Monoclonal anti-alpha-tubulin clone B-5-1-2 antibody (T5168, Sigma-Aldrich) was used as the loading control. anti-alpha-tubulinsuggested: (Sigma-Aldrich Cat# T5168, RRID:AB_477579)For phospho-protein quantification, the membranes were stripped, blocked and reprobed using a solution containing the corresponding anti-fosfospecific antibody. anti-fosfospecificsuggested: NonePhosphorylated proteins densitometry values were divided by the total protein values and the α-tubulin antibody was used as the normalizer of the amount of proteins applied in the gel. α-tubulinsuggested: None… SciScore for 10.1101/2021.06.21.449284: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Monoclonal anti-alpha-tubulin clone B-5-1-2 antibody (T5168, Sigma-Aldrich) was used as the loading control. anti-alpha-tubulinsuggested: (Sigma-Aldrich Cat# T5168, RRID:AB_477579)For phospho-protein quantification, the membranes were stripped, blocked and reprobed using a solution containing the corresponding anti-fosfospecific antibody. anti-fosfospecificsuggested: NonePhosphorylated proteins densitometry values were divided by the total protein values and the α-tubulin antibody was used as the normalizer of the amount of proteins applied in the gel. α-tubulinsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cell lines, SARS-CoV-2 and infection assays: Vero cell line (ATCC CCL-81) were maintained in DMEM medium supplemented with 10% (v/v Verosuggested: None(GenBank accession number MT126808) 20 was used to infect Vero CCL-81 and Calu-3 cells with multiplicity of infection (MOI) of 0.02. Calu-3suggested: KCLB Cat# 30055, RRID:CVCL_0609)Software and Algorithms Sentences Resources Graphics and SEM were done using GraphPad Prism software version 8.1 GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPad Software, San Diego, USA). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository 28 PRIDEsuggested: (Pride-asap, RRID:SCR_012052)Database Search and Statistical Analysis: Raw data were searched using Proteome Discoverer computational platform v2.3.0.523 (PD) using the Sequest search engine. Proteome Discoverersuggested: (Proteome Discoverer, RRID:SCR_014477)Bioinformatics analysis: Gene ontology (GO) was performed using the g: profiler tool and GOplot package 30, available in Bioconductor. GOplotsuggested: NoneBioconductorsuggested: (Bioconductor, RRID:SCR_006442)Enriched pathways were determined by Reactome and KEGG platform (q-value < 0.05)32; complementary analyses were performed using the ReactomeFIPlugIn app 31. KEGGsuggested: (KEGG, RRID:SCR_012773)Complementary analyses were performed using Perseus, Perseussuggested: (Perseus, RRID:SCR_015753)ggplot2 package, Graphpad prism v.8, and RStudio software. ggplot2suggested: (ggplot2, RRID:SCR_014601)RStudiosuggested: (RStudio, RRID:SCR_000432)Peptides identified through MS data analysis were searched in the protein to better visualize their regions using PyMOL 2.4.1 Western blotting: Cells were lysed in SDC buffer containing protease (Roche, Basel, Switzerland) and phosphatase (Sigma-Aldrich) inhibitor cocktails. PyMOLsuggested: (PyMOL, RRID:SCR_000305)The volume density of the chemiluminescent bands was calculated as integrated optical density × mm2 using ImageJ Fiji. ImageJsuggested: (ImageJ, RRID:SCR_003070)RNA-seq data reanalysis: The fastq files were downloaded from the https://sra-explorer.info/ platform with the BioProject accession number PRJNA646224 37 and processed on the Galaxy server 38. BioProjectsuggested: (NCBI BioProject, RRID:SCR_004801)The ‘FastQC’ module was used to report the quality reads, following by the trimmed using Trim Galore (v. 0.4.3.1) set to the single-end library. Trim Galoresuggested: (Trim Galore, RRID:SCR_011847)The Trim Galore output sequences were aligned to the human reference genome hg38 using the HISAT2 (Galaxy Version 2.1.0+galaxy7 Galaxysuggested: (Galaxy, RRID:SCR_006281)The differently regulated genes were analyzed by the limma, Glimma, edgeR, and Homo.sapiens packages applying a cut-off of |log2FC|>1 and a p-adjusted value <0.05 (Benjamini-Hochberg). limmasuggested: (LIMMA, RRID:SCR_010943)Glimmasuggested: (Glimma, RRID:SCR_017389)edgeRsuggested: (edgeR, RRID:SCR_012802)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 23. 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.
Results from scite Reference Check: We found no unreliable references.
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