Tunneling nanotubes provide a route for SARS-CoV-2 spreading
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Abstract
Neurological manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection represent a major issue in long coronavirus disease. How SARS-CoV-2 gains access to the brain and how infection leads to neurological symptoms are not clear because the principal means of viral entry by endocytosis, the angiotensin-converting enzyme 2 receptor, are barely detectable in the brain. We report that human neuronal cells, nonpermissive to infection through the endocytic pathway, can be infected when cocultured with permissive infected epithelial cells. SARS-CoV-2 induces the formation of tunneling nanotubes (TNTs) and exploits this route to spread to uninfected cells. In cellulo correlative fluorescence and cryo–electron tomography reveal that SARS-CoV-2 is associated with TNTs between permissive cells. Furthermore, multiple vesicular structures such as double-membrane vesicles, sites of viral replication, are observed inside TNTs between permissive and nonpermissive cells. Our data highlight a previously unknown mechanism of SARS-CoV-2 spreading, likely used as a route to invade nonpermissive cells and potentiate infection in permissive cells.
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SciScore for 10.1101/2021.11.15.468633: (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 Quantifications were done blind. Power Analysis not detected. Cell Line Authentication Authentication: After 48 h, cells expressing mCherry have been validated. Table 2: Resources
Antibodies Sentences Resources After washing, the cells were incubated 2 minutes in 0.05% PBS-tween and then incubated with the primary antibody, a polyclonal SARS-CoV-anti-N IgG, provided by Nicolas Escriou, Institut Pasteur, Paris (or alternatively with a Human SARS-CoV-2 anti-S IgG provided by Cyril Planchais from the group of Hugo Mouquet Institut Pasteur, Paris), overnight at 4 degrees. IgG provided by Cyril Planchais from the group …SciScore for 10.1101/2021.11.15.468633: (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 Quantifications were done blind. Power Analysis not detected. Cell Line Authentication Authentication: After 48 h, cells expressing mCherry have been validated. Table 2: Resources
Antibodies Sentences Resources After washing, the cells were incubated 2 minutes in 0.05% PBS-tween and then incubated with the primary antibody, a polyclonal SARS-CoV-anti-N IgG, provided by Nicolas Escriou, Institut Pasteur, Paris (or alternatively with a Human SARS-CoV-2 anti-S IgG provided by Cyril Planchais from the group of Hugo Mouquet Institut Pasteur, Paris), overnight at 4 degrees. IgG provided by Cyril Planchais from the group of Hugo Mouquet Institut Pasteur , Paris) , overnight at 4 degreessuggested: NoneAfter washing the cells were then incubated with an anti-rabbit (or an anti-human) HRP-conjugated antibody for 1 hour. anti-humansuggested: NoneThe primary antibody used were: a rabbit anti-Nucleoprotein (Anti-N, gift from Nicolas Escriou, Institut Pasteur, Paris) (1:500) over night (ON); an anti-human anti-Spike (H2-162, produced by Cyril Planchais from the group of Hugo Mouquet Institut Pasteur, Paris) (1: 100) ON, an anti-mouse J2 (1:50) (Scicons) ON, an anti-sheep nsp3 (1:200) (MRC PPU Reagents). anti-Nucleoproteinsuggested: (Bio X Cell Cat# BE0106, RRID:AB_10949017)Anti-N , gift from Nicolas Escriou , Institut Pasteur , Parissuggested: Noneanti-human anti-Spike ( H2-162suggested: Noneanti-mouse J2suggested: Noneanti-sheep nsp3suggested: (Novus Cat# NB110-61650, RRID:AB_2291744)The day after, cells were thoroughly washed and incubated for 40 min with an anti-rabbit Alexa-Fluor 633-conjugated secondary antibody (Invitrogen), an anti-human Alexa-Fluor 488-conjugated secondary antibody (Invitrogen), goat anti-mouse Alexa-Fluor 633-conjugated secondary antibody (Invitrogen) at 1:500 in 2% BSA (w/v) in PBS 1X respectively. anti-rabbitsuggested: (GenWay Biotech Inc. Cat# GWB-D633F6, RRID:AB_10283907)anti-human Alexa-Fluor 488-conjugated secondary antibody ( Invitrogen)suggested: Noneanti-mousesuggested: (GenWay Biotech Inc. Cat# GWB-F633A0, RRID:AB_10279445)For correlative light-and cryo-electron microscopy using the anti-S primary antibody, cells were fixed with PFA 4% for 15 min at 37 °C, quenched with 50 mM NH4Cl for 15 min, and blocked with PBS containing 2% BSA (w/v) for ON at 4 °C. anti-Ssuggested: NoneCells were labeled with an anti-human AlexaFluor 488-conjugated secondary antibody (Invitrogen) at 1:500 and labelled with HCS Cell Mask TM Blue Stain (Invitrogen, 1:300). anti-human AlexaFluor 488-conjugated secondary antibody ( Invitrogen )suggested: NoneExperimental Models: Cell Lines Sentences Resources The cell-lines used in this assay included Caco-2, CAD, SH-SY5Y and Vero E6. Caco-2suggested: None250 μl of each dilution was used to infect a confluent monolayer of Vero E6 cells, in a 24 wells multiwell plate, for a total of 6 wells per sample. Vero E6suggested: NoneLentiviral Transduction: Transduction of SH-SY5Y and Vero E6 cells with a lentiviral vector expressing pCMV-mcherry: 600.000 SH-SY5Y cells 400.000 Vero-E6 were plated in 60 mm plates. SH-SY5Ysuggested: NoneTo evaluate the possibility of SARS-CoV-2 transfer from donor to acceptor cells mediated by secretion, the supernatants from SARS-CoV-2 infected Vero-E6 cells were collected centrifuged at 1000 rpm for 10 min to remove floating cells and added on acceptor cells: SH-SY5Y mCherry. Vero-E6suggested: NoneRecombinant DNA Sentences Resources Lentiviral Transduction: Transduction of SH-SY5Y and Vero E6 cells with a lentiviral vector expressing pCMV-mcherry: 600.000 SH-SY5Y cells 400.000 Vero-E6 were plated in 60 mm plates. pCMV-mcherrysuggested: NoneTransduction of SH-SY5Y cells with a lentiviral vector expressing pCMV-H2B-GFP: 600.000 SH-SY5Y cells were plated in 60 mm plates. pCMV-H2B-GFPsuggested: NoneSoftware and Algorithms Sentences Resources The whole cellular volume was imaged by acquiring 0.45 µm Z-stacks with an inverted confocal microscope (Zeiss LSM 700) using ZEN software. ZENsuggested: NoneThe 3D rendering of TNTs were performed using IMARIS software. IMARISsuggested: (Imaris, RRID:SCR_007370)Statistical analysis: All column graphs and statistical analysis were performed by using GraphPad Prism version 7 software. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)PCC was calculated by using JACoP plugins 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 53, 39 and 31. 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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