Related Experiment Video

Updated: Aug 7, 2025

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Published on: May 21, 2018

SARS-COV-2 viroporins activate the NLRP3-inflammasome by the mitochondrial permeability transition pore

Joseph W Guarnieri¹, Alessia Angelin¹, Deborah G Murdock¹

¹Center for Mitochondrial and Epigenomic Medicine, Division of Human Genetics, The Children's Hospital of Philadelphia, Philadelphia, PA, United States.

Frontiers in Immunology

|March 9, 2023

Abstract

Background:

Compared to healthy controls, severe COVID19 patients display increased levels of activated NLRP3-inflammasome (NLRP3-I) and interleukin (IL)-1β. SARS-CoV-2 encodes viroporin proteins E and Orf3a(2-E+2-3a) with homologs to SARS-CoV-1, 1-E+1-3a, which elevate NLRP3-I activation; by an unknown mechanism. Thus, we investigated how 2-E+2-3a activates the NLRP3-I to better understand the pathophysiology of severe COVID-19.

Methods:

We generated a polycistronic expression-vector co-expressing 2-E+2-3a from a single transcript. To elucidate how 2-E+2-3a activates the NLRP3-I, we reconstituted the NLRP3-I in 293T cells and used THP1-derived macrophages to monitor the secretion of mature IL-1β. Mitochondrial physiology was assessed using fluorescent microscopy and plate reader assays, and the release of mitochondrial DNA (mtDNA) was detected from cytosolic-enriched fractions using Real-Time PCR.

Results:

Expression of 2-E+2-3a in 293T cells increased cytosolic Ca++ and elevated mitochondrial Ca++, taken up through the MCUi11-sensitive mitochondrial calcium uniporter. Increased mitochondrial Ca++ stimulated NADH, mitochondrial reactive oxygen species (mROS) production and the release of mtDNA into the cytosol. Expression of 2-E+2-3a in NLRP3-I reconstituted 293T cells and THP1-derived macrophages displayed increased secretion of IL-1β. Increasing mitochondrial antioxidant defenses via treatment with MnTBAP or genetic expression of mCAT abolished 2-E+2-3a elevation of mROS, cytosolic mtDNA levels, and secretion of NLRP3-activated-IL-1β. The 2-E+2-3a-induced release of mtDNA and the secretion of NLRP3-activated-IL-1β were absent in cells lacking mtDNA and blocked in cells treated with the mitochondrial-permeability-pore(mtPTP)-specific inhibitor NIM811.

Conclusion:

Our findings revealed that mROS activates the release of mitochondrial DNA via the NIM811-sensitive mitochondrial-permeability-pore(mtPTP), activating the inflammasome. Hence, interventions targeting mROS and the mtPTP may mitigate the severity of COVID-19 cytokine storms.

Insights

Severe COVID-19 involves elevated NLRP3-inflammasome (NLRP3-I) and IL-1β. SARS-CoV-2 proteins 2-E+2-3a activate NLRP3-I via mitochondrial calcium, mROS, and mtDNA release through the mtPTP.

Area of Science:

Immunology
Virology
Cell Biology

Background:

Severe COVID-19 is associated with increased NLRP3-inflammasome (NLRP3-I) activation and interleukin (IL)-1β levels compared to healthy controls.
SARS-CoV-2 viroporin proteins 2-E+2-3a are homologous to SARS-CoV-1 proteins and are known to activate NLRP3-I through an uncharacterized mechanism.

Purpose of the Study:

To investigate the mechanism by which SARS-CoV-2 proteins 2-E+2-3a activate the NLRP3-inflammasome.
To elucidate the role of mitochondrial dysfunction in COVID-19 pathophysiology.

Main Methods:

Co-expression of SARS-CoV-2 proteins 2-E+2-3a using a polycistronic vector.
Reconstitution of the NLRP3-inflammasome in 293T cells and use of THP1-derived macrophages to assess IL-1β secretion.
Evaluation of mitochondrial physiology, including calcium uptake, reactive oxygen species (mROS) production, and mitochondrial DNA (mtDNA) release.

Main Results:

Expression of 2-E+2-3a increased cytosolic and mitochondrial calcium levels, stimulating mROS production and mtDNA release into the cytosol.
2-E+2-3a expression led to increased IL-1β secretion in NLRP3-I reconstituted cells and macrophages.
Inhibition of mROS or blocking the mitochondrial-permeability-pore (mtPTP) with NIM811 prevented 2-E+2-3a-induced mtDNA release and IL-1β secretion.

Conclusions:

Mitochondrial reactive oxygen species (mROS) activate the release of mitochondrial DNA (mtDNA) through the NIM811-sensitive mitochondrial-permeability-pore (mtPTP), subsequently activating the inflammasome.
Targeting mROS and the mtPTP presents a potential therapeutic strategy to mitigate severe COVID-19 and cytokine storms.

Read full analysis

Keywords:

COVID-19 NLRP3-inflammasome mitochondria in innate immune responses mitochondrial permeability transition pore viroporin

More Related Videos

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells

Published on: May 22, 2014

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Published on: January 20, 2023

Related Concept Videos

The Intrinsic Apoptotic Pathway

The Intrinsic Apoptotic Pathway

Internal cellular stress, such as cellular injury or hypoxia, triggers intrinsic apoptosis. The B-cell lymphoma 2 (Bcl-2) family of proteins are the primary regulators of the intrinsic apoptotic pathway. For example, during DNA damage, checkpoint proteins, such as Ataxia Telangiectasia Mutated (ATM protein) and Checkpoints Factor-2 (Chk2) proteins, are activated. These proteins phosphorylate p53 which further activates pro-apoptotic proteins, such as Bax, Bak, PUMA, and Noxa, and inhibits...

Porin Insertion in the Outer Mitochondrial Membrane

Porin Insertion in the Outer Mitochondrial Membrane

Porins are beta-barrel proteins translocated to the mitochondrial outer membrane through the TOM complex into the intermembrane space. Porin precursors bind TIM chaperones within the intermembrane space and are guided to the Sorting and Assembly Machinery complex or SAM complex on the outer mitochondrial membrane.
Three models describe the assembly of porins by the SAM complex and their insertion into the outer membrane. Model 1 suggests that porins are assembled outside the SAM channel as the...

The Extrinsic Apoptotic Pathway

The Extrinsic Apoptotic Pathway

The extrinsic apoptotic pathway is initiated when extracellular death-inducing signals, such as specific cytokines, activate the death receptors expressed on the cell surface. The immune cells involved in this pathway are natural killer cells (NK cells) and cytotoxic T-lymphocytes. NK cells are critical in innate immune response, while cytotoxic T-lymphocytes are associated with adaptive immune response. These cells recognize specific receptors expressed on the altered cells and activate...

Structure of Porins

Structure of Porins

Mitochondria, chloroplasts, and gram-negative bacteria have transmembrane, beta-barrel proteins called porins to mediate the free diffusion of ions and metabolites across the membrane. Mitochondrial porin precursors contain conserved amino acid sequences called beta signals at their C-terminal. Beta signals have a motif of PoXGXXHyXHy (Po-Polar, X-Any amino acid, G-Glycine, Hy-LargeHydrophobic), which are crucial for precursor recognition to initiate precursor assembly. Beta-barrel...

Translocation of Proteins into the Mitochondria

Translocation of Proteins into the Mitochondria

Mitochondrial precursors are translocated to the internal subcompartments via independent mechanisms involving distinct protein machineries called translocases.
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...

Protein Transport into the Inner Mitochondrial Membrane

Protein Transport into the Inner Mitochondrial Membrane

Nuclear encoded mitochondrial precursors are imported to the inner membrane in a multistep process involving two separate translocons, TIM22 and TIM23. TIM23 is a cation-selective pore that remains closed by the N terminal segment of the protein. Negative charges on the TIM23 act as a receptor for the incoming precursor, pulling the positively charged matrix-targeting sequence for peptide insertion and translocation.
Transport of mitochondrial precursors across the TIM23 channel is driven by...

Articles linked to this work by shared authors, journal, and citation graph.

Agentic AI in Medicine: Challenges for Responsible Development and the Case for Clinical Testing Harnesses.

Annals of biomedical engineering·2026

Prompt Injection in Clinical Artificial Intelligence Systems: The Emerging Security Challenge of Large Language Models and Agentic AI.

Annals of biomedical engineering·2026

Allotopic Expression of ND6 Restores Vision in a Mitochondrial Disease Model.

JCI insight·2026

AI agents in clinical medicine: delegation, supervision, and the problem of correlated failure.

Irish journal of medical science·2026

Regional Stabilization Before Transfer in Moderate Traumatic Brain Injury: Comparable Outcomes Within a Rural Hub-and-Spoke Trauma System.

The American surgeon·2026

MSeqDR PMD-VR: An Expert-Curated Virtual Registry of 11,000 Mitochondrial Disease Cases Established Through Literature Mining and Generative AI Augmentation.

Genes·2026

See all related articles