SARS-CoV-2 Viroporins Activate The NLRP3-Inflammasome Via The Mitochondrial Permeability Transition Pore
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Abstract
Cytokine storm precipitated by activation of the host innate immune defenses is a major cause of COVID19 death. To elucidate how SARS-CoV-2 initiates this inflammatory process, we studied viroporin proteins E and Orf3a (2-E+2-3a). Expression of 2-E+2-3a in human 293T cells resulted in increased cytosolic Ca ++ and then elevated mitochondrial Ca ++ , taken up through the MUCi11-sensitive mitochondrial calcium uniporter (MCU). Increased mitochondrial Ca ++ resulted in stimulation of mitochondrial reactive oxygen species (mROS) production, which was blocked by mitochondrially-targeted catalase or MnTBAP. To determined how mROS activates the inflammasome, we transformed 293T cells with NLRP3, ASC, pro-caspase-1 and pro-IL-1β plus used THP1 derived macrophages to monitor the secretion of mature IL-1β. This revealed that mROS activates a factor that is released via the NIM811-sensitive mitochondrial permeability pore (mtPTP) to activate the inflammasome. Hence, interventions targeting mROS and the mtPTP may mitigate the severity of COVID19 cytokine storms.
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SciScore for 10.1101/2022.02.19.481139: (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
Experimental Models: Cell Lines Sentences Resources Twenty-four hours post-plating sub-confluent monolayers of 293T cells were infected with LV-EV or LV-E3a. 293Tsuggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)THP-1 cells were plated at a density of 100 × 103 in 96-well plates. THP-1suggested: NoneTHP1 cells were infected with LV-EV or LV-E3a. THP1suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006)Software and Algorithms Sentences Resources Fluorescence quantified using the Zeiss 710 LSM confocal microscope was … SciScore for 10.1101/2022.02.19.481139: (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
Experimental Models: Cell Lines Sentences Resources Twenty-four hours post-plating sub-confluent monolayers of 293T cells were infected with LV-EV or LV-E3a. 293Tsuggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)THP-1 cells were plated at a density of 100 × 103 in 96-well plates. THP-1suggested: NoneTHP1 cells were infected with LV-EV or LV-E3a. THP1suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006)Software and Algorithms Sentences Resources Fluorescence quantified using the Zeiss 710 LSM confocal microscope was analyzed using ImageJ. ImageJsuggested: (ImageJ, RRID:SCR_003070)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: We detected the following sentences addressing limitations in the study:Limitations of the Study: A unique feature of this research is the discovery that SARS-CoV-2 viroporins active the inflammasome via the mitochondrion through elevated mitochondrial Ca++ and mROS and the mtPTP. However, we have not identified this released mitochondrial oxidized product. Rather, we relied of the publications of others implicating Ox-mtDNA.
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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