DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist
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Abstract
The efficient spread of SARS-CoV-2 resulted in a unique pandemic in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLR S ) of antigen-presenting cells, widely present in respiratory mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2 + Vero E6 cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. These data have been then confirmed using authentic SARS-CoV-2 virus and human respiratory cell lines. Thus, we described a mechanism potentiating viral spreading of infection.
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SciScore for 10.1101/2020.08.09.242917: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: Production of human monocyte-derived macrophages and dendritic cells: Blood samples were obtained from healthy human donors (Hospital 12 de Octubre, Madrid, Spain) under informed consent and IRB approval. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources To generate monocyte-derived macrophages (M2-MDMs), CD14+ monocytes were purified using anti-human CD14 antibody-labeled magnetic beads and iron-based LS columns (Miltenyi Biotec) and used directly for further differentiation into macrophages (Dominguez-Soto … SciScore for 10.1101/2020.08.09.242917: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: Production of human monocyte-derived macrophages and dendritic cells: Blood samples were obtained from healthy human donors (Hospital 12 de Octubre, Madrid, Spain) under informed consent and IRB approval. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources To generate monocyte-derived macrophages (M2-MDMs), CD14+ monocytes were purified using anti-human CD14 antibody-labeled magnetic beads and iron-based LS columns (Miltenyi Biotec) and used directly for further differentiation into macrophages (Dominguez-Soto et al., J Immunol 2011). anti-human CD14suggested: NoneAs a control, inhibition experiment was performed in the presence of anti-DC/L-SIGN antibody (R&D Systems). anti-DC/L-SIGNsuggested: NoneExperimental Models: Cell Lines Sentences Resources EXPI293 cells grown in EXPI293 expression medium were transiently transfected with the S ectodomain vector according to the manufacturer’s protocol (Thermo Fisher Scientific). EXPI293suggested: RRID:CVCL_D615)Cell lines: Baby hamster kidney cells (BHK-21, 12-14-17 MAW, Kerafast, Boston, MA) and African Green Monkey Cell Line (VeroE6) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 25 μg/mL gentamycin and 2 mM L-glutamine. BHK-21suggested: NoneTrans-infection: For trans-infection studies, Jurkat DC-SIGN, Jurkat L-SIGN, Jurkat Langerin (3 × 105 cells) or MDDCs (5 × 104 cells) were challenged with recombinant SARS-CoV-2, EBOV-GP or VSV-G pseudotyped viruses (MOI: 0.5-2) and incubated during 2 h at room temperature with rotation. Jurkatsuggested: NoneJurkat DC-SIGN and MDDCs were then resuspended in RPMI medium and co-cultivated with adherent Vero E6 cells (1.5 × 105 cells/well) on a 24-well plate. Jurkat DC-SIGNsuggested: NoneVero E6suggested: NoneSoftware and Algorithms Sentences Resources To facilitate the visualization of the molecules, a Gaussian filter was applied to the images using Photoshop, then the gray levels were saturated and the background eliminated. Photoshopsuggested: (Adobe Photoshop, RRID:SCR_014199)For the 2D classification, images were processed with RELION 2.1 (Scheres, 2012). RELIONsuggested: (RELION, RRID:SCR_016274)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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
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.
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SciScore for 10.1101/2020.08.09.242917: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Blood samples were obtained from healthy human donors (Hospital 12 de Octubre, Madrid, Spain) under informed consent and IRB approval. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources To generate monocyte-derived macrophages (M2-MDMs), CD14+ monocytes were purified using anti-human CD14 antibodylabeled magnetic beads and iron-based LS columns (Miltenyi Biotec) and used directly for further differentiation into macrophages (Dominguez-Soto et al., J Immunol 2011). anti-huma…SciScore for 10.1101/2020.08.09.242917: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Blood samples were obtained from healthy human donors (Hospital 12 de Octubre, Madrid, Spain) under informed consent and IRB approval. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources To generate monocyte-derived macrophages (M2-MDMs), CD14+ monocytes were purified using anti-human CD14 antibodylabeled magnetic beads and iron-based LS columns (Miltenyi Biotec) and used directly for further differentiation into macrophages (Dominguez-Soto et al., J Immunol 2011). anti-human CD14suggested: NoneAs a control, inhibition experiment was performed in the presence of anti-DC/L-SIGN antibody (R&D Systems). anti-DC/L-SIGNsuggested: NoneExperimental Models: Cell Lines Sentences Resources EXPI293 cells grown in EXPI293 expression medium were transiently transfected with the S ectodomain vector according to the manufacturer’s protocol (Thermo Fisher Scientific). EXPI293suggested: RRID:CVCL_D615)Cell lines Baby hamster kidney cells (BHK-21, 12-14-17 MAW, Kerafast, Boston, MA) and African Green Monkey Cell Line (VeroE6) were cultured in Dulbecco´s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 25 μg/mL gentamycin and 2 mM L-glutamine. BHK-21suggested: NoneJurkat, Jurkat DC-SIGN, Jurkat L-SIGN (Alvarez et al., 2002) and Jurkat langerin were maintained in RPMI 1640 supplemented with 10% heat-inactivated FBS, 25 μg/mL gentamycin and 2 mM L-glutamine. Jurkatsuggested: NoneInfectious titers were estimated as tissue culture infectious dose per mL by limiting dilution of rVSV-luc-pseudotypes on Vero E6 cells. Vero E6suggested: RRID:CVCL_XD71)Jurkat DC-SIGN and MDDCs were then resuspended in RPMI medium and co-cultivated with adherent Vero E6 cells (1.5 x 105 cells/well) on a 24-well plate. Jurkat DC-SIGNsuggested: NoneSoftware and Algorithms Sentences Resources ( Top row: original images; bottom row: Photoshop processed images. Photoshopsuggested: (Adobe Photoshop, RRID:SCR_014199)For the 2D classification, images were processed with RELION 2.1 (Scheres, 2012). RELIONsuggested: (RELION, RRID:SCR_016274)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 Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
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