SARS-CoV-2 proteins bind heme and hemoglobin
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Abstract
The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome virus 2 (SARS-CoV-2), has led to a global crisis that included collapsing healthcare systems and shut-down communities, producing considerable economic burden. Despite the number of effective vaccines quickly implemented, the emergence of new variants is a primary concern. The scientific community undertook a rapid response to better study this new virus. However, critical questions about viral protein-protein interactions and mechanisms of its physiopathology are still unclear. Although severe COVID-19 was associated with hematological dysfunctions, scarce experimental data were produced about iron dysmetabolism and the viral proteins’ possible interaction with hemoglobin (Hb) chains. This work demonstrates the binding of SARS-CoV-2 proteins to hemin and Hb using a multimethodological approach. In silico analysis indicated binding motifs between a cavity in the viral nucleoprotein and hemoglobin’s porphyrin coordination region. Different hemin binding capacities of mock and SARS-CoV-2-infected culture extracts were noticed using gel electrophoresis and TMB staining. Hemin-binding proteins were isolated from SARS-CoV-2-infected cells by affinity chromatography and identified by shotgun proteomics, indicating that structural (nucleoprotein, spike, and membrane protein) and non-structural (Nsp3 and Nsp7) viral proteins interact with hemin. In vitro analyses of virus adsorption to host cells and viral replication studies in Vero cells demonstrated inhibitory activities - at different levels - by hemin, protoporphyrin IX (PpIX) Hb. Strikingly, free Hb at 1μM suppressed viral replication (99 %), and its interaction with SARS-CoV-2 was localized to the RBD region of the Spike protein. The findings showed clear evidence of new avenues to disrupt viral replication and understand virus physiopathology that warrants further investigation.
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SciScore for 10.1101/2021.04.16.440124: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. 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 Then, membranes were washed and incubated with HRP-conjugated anti-human IgG antibody (Sigma-Aldrich; 1:10.000) followed by chemiluminescence detection. anti-human IgGsuggested: NoneAfter washing with TBS-T, the membrane was incubated with an anti-Hb antibody (1:5.000) (Sigma-Aldrich). anti-Hbsuggested: NoneAfter incubation, membranes were washed with TBS-T, followed by additional incubation with anti-human Hb antibody for 90 min. anti-human Hbsuggested: NoneSciScore for 10.1101/2021.04.16.440124: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. 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 Then, membranes were washed and incubated with HRP-conjugated anti-human IgG antibody (Sigma-Aldrich; 1:10.000) followed by chemiluminescence detection. anti-human IgGsuggested: NoneAfter washing with TBS-T, the membrane was incubated with an anti-Hb antibody (1:5.000) (Sigma-Aldrich). anti-Hbsuggested: NoneAfter incubation, membranes were washed with TBS-T, followed by additional incubation with anti-human Hb antibody for 90 min. anti-human Hbsuggested: NoneSubsequently, the membrane was washed with TBS-T and incubated for 90 min with anti-rabbit IgG antibody conjugated to alkaline phosphatase (Sigma Alrich), diluted 1:5.000 in TBS-T solution containing 0.75 % BSA. anti-rabbit IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Alternatively, two experimental conditions were performed: i) preincubation of the virus with the compounds (1 μM) for 1 h at 37 °C before their addition to Vero E6 cells (MOI of 0.01) for an additional hour, or ii) preincubation of Vero cells with the compounds (1 μM) for 1 h at 37 °C before their exposure to the virus (MOI 0.01). Vero E6suggested: NoneVerosuggested: NoneSoftware and Algorithms Sentences Resources Data were acquired using Xcalibur software (version 3.0.63). Xcalibursuggested: (Thermo Xcalibur, RRID:SCR_014593)Data analysis: Graphs were prepared, and statistics were performed using R software 4.0.0 and GraphPad Prism version 9. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)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 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.
- No funding statement was detected.
- No protocol registration statement was detected.
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