Endomembrane systems are reorganized by ORF3a and Membrane (M) of SARS-CoV-2
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Abstract
The endomembrane reticulum (ER) is largely reorganized by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 ORF3a and membrane (M) protein expression affects ER-derived structures including cubic membrane and double membrane vesicles in coronavirus-infected cells; however, the molecular mechanisms underlying ER remodeling remain unclear. We introduced a “plug and playable” proximity labeling tool (TurboID-GBP) for interactome mapping of GFP-tagged SARS-CoV-2 ORF3a and M proteins. Through mass spectrometric identification of the biotinylated lysine residue (K+226 Da) on the viral proteins using Spot-TurboID workflow, 117 and 191 proteins were robustly determined as ORF3a and M interactomes, respectively, and many, including RNF5 (E3 ubiquitin ligase), overlap with the mitochondrial-associated membrane (MAM) proteome. RNF5 expression was correlated to ORF3a ubiquitination. MAM formation and secreted proteome profiles were largely affected by ORF3a expression. Thus, SARS-CoV-2 may utilize MAM as a viral assembly site, suggesting novel anti-viral treatment strategies for blocking viral replication in host cells.
Highlights
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SARS-CoV-2 proteins ORF3a and M alter endoplasmic reticulum proteome profile
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ORF3a affects mitochondrial-associated membrane formation
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SARS-CoV-2 may utilize mitochondrial-associated membrane as viral assembly site
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ORF3a and M interactome proteins may serve as targets for COVID-19 treatment
eTOC Blurb
ER remodelling by SARS-CoV-2 ORF3a and M protein
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SciScore for 10.1101/2021.06.01.446555: (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 To detect expression pattern or biotinylation pattern, the primary antibody, such as anti-V5 (Invitrogen, cat. No. R960-25, 1:5,000 dilution), was incubated for 1 h at room temperature. anti-V5suggested: (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564)After washing three times with TBST for 5 min at room temperature, secondary antibodies, namely mouse-HRP (Bio-rad, cat. No. 1706516, 1:3,000 dilution) and rabbit-HRP (Cell signaling, cat. No. 7074S, 1:3,000 dilution), or SA-HRP … SciScore for 10.1101/2021.06.01.446555: (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 To detect expression pattern or biotinylation pattern, the primary antibody, such as anti-V5 (Invitrogen, cat. No. R960-25, 1:5,000 dilution), was incubated for 1 h at room temperature. anti-V5suggested: (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564)After washing three times with TBST for 5 min at room temperature, secondary antibodies, namely mouse-HRP (Bio-rad, cat. No. 1706516, 1:3,000 dilution) and rabbit-HRP (Cell signaling, cat. No. 7074S, 1:3,000 dilution), or SA-HRP (Thermofisher, cat. No. 21126, 1:10,000 dilution) were incubated for 1 h at room temperature. mouse-HRPsuggested: Nonerabbit-HRPsuggested: (Carl Roth Cat# 4750, RRID:AB_2890006)Experimental Models: Cell Lines Sentences Resources HeLa cells and A549 cells were from Laboratory of Dr. HeLasuggested: NoneA549suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)Obtaining Secreted Proteins in Cell Culture Media: After biotin labeling of HEK293 cells expressing SEC61B-TurboID, cells were incubated with no FBS DMEM for 16 h. HEK293suggested: NoneProteome Digestion & Enrichment of Biotinylated Peptides: For mass sampling, HEK293T cells were split in three T75 flasks for triplicate samples per each condition. HEK293Tsuggested: NoneRecombinant DNA Sentences Resources Expression Plasmids: Genes with epitope tag (i.e. V5, FLAG, HA, twin strep) were cloned into pCDNA3, pCDNA3.1, and pCDNA5 using digestion with enzymatic restriction sites and ligation with T4 DNA ligase. pCDNA3suggested: RRID:Addgene_15475)pCDNA3.1suggested: RRID:Addgene_79663)pCDNA5suggested: RRID:Addgene_49428)For transiently co-expressed two constructs, vPOI-GFP and TurboID-GBP, cells were grown at 70–80% confluence and transfected with wanted constructs using PEI. vPOI-GFPsuggested: NoneSoftware and Algorithms Sentences Resources Pearson correlation was conducted using image J software program (NIH). image Jsuggested: (ImageJ, RRID:SCR_003070)After washing three times with TBST for 5 min at room temperature, results of immunoblotting assay were obtained using ECL solution using GENESYS program. GENESYSsuggested: (GeneSys, RRID:SCR_015770)Enlarged fluorescence images were fitted to the electron micrographs using the Image J BigWarp program. Image J BigWarpsuggested: NoneProcessing MS data and Identification of Proteins: All MS/MS data were searched using MaxQuant (version 1.5.3.30) with Andromeda search engine at 10 ppm precursor ion mass tolerance against the SwissProt Homo sapiens proteome database (20,199 entries, UniProt (http://www.uniprot.org/)). MaxQuantsuggested: (MaxQuant, RRID:SCR_014485)UniProtsuggested: (UniProtKB, RRID:SCR_004426)The imputation of protein intensity were conducted using Perseus software program. Perseussuggested: (Perseus, RRID:SCR_015753)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: We did not find any issues relating to colormaps.
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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