{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,19]],"date-time":"2026-10-19T23:33:42Z","timestamp":1792452822813,"version":"4.5.0"},"reference-count":52,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,2,20]],"date-time":"2023-02-20T00:00:00Z","timestamp":1676851200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000005","name":"U.S. Department of Defense","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000005","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p>Compared to healthy controls, severe COVID19 patients display increased levels of activated NLRP3-inflammasome (NLRP3-I) and interleukin (IL)-1\u03b2. 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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>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-1\u03b2. 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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>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-1\u03b2. 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-1\u03b2. The 2-E+2-3a-induced release of mtDNA and the secretion of NLRP3-activated-IL-1\u03b2 were absent in cells lacking mtDNA and blocked in cells treated with the mitochondrial-permeability-pore(mtPTP)-specific inhibitor NIM811.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2023.1064293","type":"journal-article","created":{"date-parts":[[2023,2,20]],"date-time":"2023-02-20T05:48:55Z","timestamp":1676872135000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":62,"title":["SARS-COV-2 viroporins activate the NLRP3-inflammasome by the mitochondrial permeability transition pore"],"prefix":"10.3389","volume":"14","author":[{"given":"Joseph W.","family":"Guarnieri","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alessia","family":"Angelin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Deborah 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